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Trends in Pharmacological Sciences
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Salt-inducible kinases: new players in pulmonary arterial hypertension?

  • Tatiana António
    Affiliations
    Department of Biomedicine, Unit of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal

    MedInUP – Center for Drug Discovery and Innovative Medicines, University of Porto, Porto, Portugal
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  • Patrício Soares-da-Silva
    Affiliations
    Department of Biomedicine, Unit of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal

    MedInUP – Center for Drug Discovery and Innovative Medicines, University of Porto, Porto, Portugal
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  • Nuno M. Pires
    Affiliations
    Department of Research, BIAL-Portela & Cª, S.A., Coronado, Portugal
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  • Pedro Gomes
    Correspondence
    Correspondence:
    Affiliations
    Department of Biomedicine, Unit of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal

    Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Coimbra, Portugal
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      Highlights

      • Salt-inducible kinases (SIK1–3) are serine/threonine protein kinases that target several effector molecules implicated in the regulation of a variety of cellular processes including metabolism, inflammation, and cancer.
      • Recent advances in the mechanistic understanding of pulmonary arterial hypertension (PAH) suggest a critical role for deregulated signaling pathways in patients and animal models.
      • Multiple lines of evidence support functional parallels between SIK-regulated pathways in tumorigenesis, inflammation, and PAH.
      • A better understanding of SIK functions and downstream targets may unlock the potential of SIKs as promising therapeutic targets for the development of much-needed therapies for PAH.
      Salt-inducible kinases (SIKs) are serine/threonine kinases belonging to the AMP-activated protein kinase (AMPK) family. Accumulating evidence indicates that SIKs phosphorylate multiple targets, including histone deacetylases (HDACs) and cAMP response element-binding protein (CREB)-regulated transcriptional coactivators (CRTCs), to coordinate signaling pathways implicated in metabolism, cell growth, proliferation, apoptosis, and inflammation. These pathways downstream of SIKs are altered not only in pathologies like cancer, systemic hypertension, and inflammatory diseases, but also in pulmonary arterial hypertension (PAH), a multifactorial disease characterized by pulmonary vasoconstriction, inflammation and remodeling of pulmonary arteries owing to endothelial dysfunction and aberrant proliferation of smooth muscle cells (SMCs). In this opinion article, we present evidence of SIKs as modulators of key signaling pathways involved in PAH pathophysiology and discuss the potential of SIKs as therapeutic targets for PAH, emphasizing the need for deeper molecular insights on PAH.

      Keywords

      PAH: the basics

      PAH is a chronic and rare disease, estimated to affect 11–60 adults per million worldwide [
      • Butrous G.
      Pulmonary hypertension: from an orphan disease to a global epidemic.
      ]. An extensive range of causes may underlie PAH pathogenesis, on the basis of which PAH is classified as idiopathic, heritable, drug-induced, disease-associated, and other subtypes. Regardless of the etiology, PAH results from the combination of sustained vasoconstriction, inflammation, and remodeling of the distal pulmonary arteries, associated with aberrant proliferation of both endothelial cells (ECs) and SMCs (Figure 1) [
      • Kurakula K.
      • et al.
      Endothelial dysfunction in pulmonary hypertension: cause or consequence?.
      ,
      • Schermuly R.T.
      • et al.
      Mechanisms of disease: pulmonary arterial hypertension.
      ,
      • Lau E.M.T.
      • et al.
      Epidemiology and treatment of pulmonary arterial hypertension.
      ]. These vascular alterations are primarily caused by endothelial dysfunction that disrupts the fine-tuned balance between vasodilators/growth inhibitors and vasoconstrictors/prothrombotic/proliferative mediators [
      • Schermuly R.T.
      • et al.
      Mechanisms of disease: pulmonary arterial hypertension.
      ,
      • Lau E.M.T.
      • et al.
      Epidemiology and treatment of pulmonary arterial hypertension.
      ,
      • Grippi M.A.
      • et al.
      Fishman’s pulmonary diseases and disorders, 2-volume set.
      ]. Major hallmarks of PAH include the abnormal release of cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF-α), that sustain a chronic inflammatory environment [
      • Hemnes A.R.
      • Humbert M.
      Pathobiology of pulmonary arterial hypertension: understanding the roads less travelled.
      ,
      • Vaillancourt M.
      • et al.
      Adaptation and remodelling of the pulmonary circulation in pulmonary hypertension.
      ], and the upregulation of numerous growth factors, importantly transforming growth factor beta (TGF-β) (Figure 1) [
      • Hemnes A.R.
      • Humbert M.
      Pathobiology of pulmonary arterial hypertension: understanding the roads less travelled.
      ,
      • Vaillancourt M.
      • et al.
      Adaptation and remodelling of the pulmonary circulation in pulmonary hypertension.
      ,
      • Ranchoux B.
      • et al.
      Endothelial dysfunction in pulmonary arterial hypertension: an evolving landscape (2017 Grover Conference Series).
      ].
      Figure 1
      Figure 1Vascular remodeling and key features of pulmonary arterial hypertension (PAH) pathophysiology.
      Diagram showing pulmonary vascular remodeling in PAH, which results in narrowing or complete obstruction of the vessel lumen, further elevating arterial pressure in the pulmonary circulation and increasing the load on the right ventricle. The disease triggers and the molecular and histological aspects most relevant in current research on PAH therapeutics are shown below. Abbreviations: BMPR2, bone morphogenetic protein receptor 2; CAV1, caveolin-1; CTD, connective tissue disorder; KCNK3, potassium channel subfamily K member 3; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor beta; VEGF, vascular endothelial growth factor. Figure generated with BioRender (https://biorender.com/).
      Similarly to cancer, proliferating cells may also originate from ECs transitioning into a mesenchymal phenotype [endothelial-to-mesenchymal transition (EndMT)] (see Glossary) [
      • Nikitopoulou I.
      • et al.
      Vascular endothelial-cadherin downregulation as a feature of endothelial transdifferentiation in monocrotaline-induced pulmonary hypertension.
      ]. TWIST1, SNAIL, and SLUG are important transcription factors that initiate EndMT and are overexpressed in the lungs of experimental models and patients with PAH [
      • Ranchoux B.
      • et al.
      Endothelial dysfunction in pulmonary arterial hypertension: an evolving landscape (2017 Grover Conference Series).
      ,
      • Stenmark K.R.
      • et al.
      Endothelial-to-mesenchymal transition: an evolving paradigm and a promising therapeutic target in PAH.
      ].
      The progressive narrowing of pulmonary arteries increases the circulatory flow resistance and pulmonary arterial pressure [
      • Lau E.M.T.
      • et al.
      Epidemiology and treatment of pulmonary arterial hypertension.
      ,
      • Simonneau G.
      • et al.
      Haemodynamic definitions and updated clinical classification of pulmonary hypertension.
      ,
      • Montani D.
      • et al.
      Pulmonary arterial hypertension.
      ,
      • Gelzinis T.A.
      Pulmonary hypertension in 2021: part I – definition, classification, pathophysiology, and presentation.
      ], placing an overload on the right ventricle (RV) of the heart, thus leading to increased RV contractility and wall thickness to cope with the elevated RV pressure. The resulting hypertrophy, high metabolic demand and reduced output eventually lead to RV failure [
      • Grippi M.A.
      • et al.
      Fishman’s pulmonary diseases and disorders, 2-volume set.
      ,
      • Montani D.
      • et al.
      Pulmonary arterial hypertension.
      ,
      • Gelzinis T.A.
      Pulmonary hypertension in 2021: part I – definition, classification, pathophysiology, and presentation.
      ].
      Despite the intensive search for PAH-specific treatment strategies, the disease remains incurable. Current therapies mainly target pulmonary vasoconstriction and provide some degree of symptomatic relief, but fail to address other underlying pathological processes, which translates into poor survival rates. It is now evident that treating vasoconstriction alone is insufficient, and novel therapeutic options need to additionally target vascular remodeling and inflammation to improve long-term survival.
      Considering the prominent role of SIKs in pathologies that share common molecular mechanisms with PAH, we propose that deregulated SIK pathways may explain disease predisposition and provide a new therapeutic avenue. In this opinion article, we highlight recent evidence of SIKs as modulators of signaling pathways relevant in the pathophysiology of PAH and discuss emerging breakthroughs that suggest the potential of SIKs as therapeutic targets for the management of PAH.

      SIKs in vasoconstriction, remodeling, and inflammation

      SIKs are serine/threonine kinases belonging to the family of AMPKs [
      • Sjöström M.
      • et al.
      SIK1 is part of a cell sodium-sensing network that regulates active sodium transport through a calcium-dependent process.
      ,
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ]. Three isoforms (SIK1, SIK2, and SIK3) have been identified (Box 1), which have both redundant and distinct physiological roles. While SIK1 mRNA expression is upregulated by membrane depolarization, adrenocorticotropic hormone, glucagon, and circadian rhythm, SIK2 and SIK3 modulation is less clear. SIKs regulate several metabolic processes, including gluconeogenesis, lipogenesis, adipogenesis, and steroidogenesis [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Wein M.N.
      • et al.
      Salt-inducible kinases: physiology, regulation by cAMP, and therapeutic potential.
      ,
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ,
      • Chen F.
      • et al.
      Salt-inducible kinase 2: an oncogenic signal transmitter and potential target for cancer therapy.
      ,
      • Yong Kim S.
      • et al.
      Salt-inducible kinases 1 and 3 negatively regulate Toll-like receptor 4-mediated signal.
      ]. Their pathological functions, however, are less well studied, but research over the past 15 years has established a role for SIKs in multiple human diseases, such as hypertension, inflammatory bowel diseases, osteoarthritis and osteoporosis, asthma, depression, sleep dysregulation, epilepsy, diabetes, and even cancer [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Wein M.N.
      • et al.
      Salt-inducible kinases: physiology, regulation by cAMP, and therapeutic potential.
      ,
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ,
      • Pires N.M.
      • et al.
      Acute salt loading induces sympathetic nervous system overdrive in mice lacking salt-inducible kinase 1 (SIK1).
      ,
      • Medina C.B.
      • et al.
      Pannexin 1 channels facilitate communication between T cells to restrict the severity of airway inflammation.
      ].
      Structure and upstream regulators of SIKs
      Three isoforms of SIKs have been identified and all appear to be expressed ubiquitously by vertebrates. Like most AMPK-related kinases, SIK isoforms share a conserved structure (Figure I) comprising an N-terminal domain with a LKB1 phosphorylation site, crucial for their catalytic activity, and a C-terminal domain with multiple PKA phosphorylation sites. Since LKB1 is constitutively active, it is widely accepted that SIKs are also constitutively phosphorylated and in an active state [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Sakamoto K.
      • et al.
      The salt-inducible kinases: emerging metabolic regulators.
      ]. SIKs may also be phosphorylated by PKA, which promotes their interaction with 14-3-3 scaffold proteins that block access to substrates, therefore resulting in an inhibitory effect [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ,
      • Takemori H.
      • et al.
      ACTH-induced nucleocytoplasmic translocation of salt-inducible kinase. Implication in the protein kinase A-activated gene transcription in mouse adrenocortical tumor cells.
      ]. Since SIK2 has more PKA phosphorylation sites than the other isoforms, this may favor its inhibition by PKA-activating agents [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Wein M.N.
      • et al.
      Salt-inducible kinases: physiology, regulation by cAMP, and therapeutic potential.
      ]. Because many hormones use cAMP as second messenger in intracellular signaling transduction, the ability of SIKs to control gene expression in response to extracellular metabolic cues might be one of their major roles. As such, their role in modulating GPCR signaling may be of great significance for therapeutic applications.
      SIKs are of particular interest because, unlike other AMPK-related kinases, they can be activated by calcium-dependent protein kinase (CaMK)-mediated phosphorylation. This implies that SIKs are sensitive to sodium and calcium imbalances, which makes them central in regulatory ion networks [
      • Butrous G.
      Pulmonary hypertension: from an orphan disease to a global epidemic.
      ,
      • Kurakula K.
      • et al.
      Endothelial dysfunction in pulmonary hypertension: cause or consequence?.
      ].
      Figure I
      Figure ISalt-inducible kinases (SIKs) as members of the family of AMP-activated protein kinase (AMPK)-related kinases phosphorylated by liver kinase B1 (LKB1) and respective domain structures.
      (A) Family of kinases phosphorylated and activated by LKB1, in which SIKs are included. (B) Schematic of the domain structure and phosphorylation sites of human SIK1 (Uniprot P57059), SIK2 (Uniprot Q9H0K1), and SIK3 (Uniprot Q9Y2K2) depicting the N-terminal kinase domain containing the phosphorylation site for LKB1, the ubiquitin-associated (UBA) domain, and the C-terminal domain containing multiple phosphorylation sites for protein kinase A (PKA). Abbreviation: CaMK, calcium-dependent protein kinase.
      The pharmacological potential of SIKs is demonstrated by accumulating evidence confirming their ability to coordinate cell growth, proliferation, and apoptosis resistance mechanisms, inflammatory responses, and extracellular matrix remodeling [
      • Medina C.B.
      • et al.
      Pannexin 1 channels facilitate communication between T cells to restrict the severity of airway inflammation.
      ,
      • Cai Y.
      • et al.
      Salt-inducible kinase 3 promotes vascular smooth muscle cell proliferation and arterial restenosis by regulating AKT and PKA–CREB signaling.
      ,
      • Hutchinson L.D.
      • et al.
      Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses.
      ,
      • Bertorello A.M.
      • et al.
      Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).
      ]. This owes to the fact that SIKs participate in numerous metabolic pathways by directly phosphorylating and controlling the subcellular localization of specific substrates.
      The best-characterized SIK substrates are two classes of transcriptional regulators: class IIa HDACs (HDAC4, HDAC5, HDAC7, and HDAC9) and CRTCs (CRTC1, CRTC2, and CRTC3) [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ]. When phosphorylated, these substrates bind 14-3-3 scaffold proteins and are retained in the cytoplasm, unable to bind and activate the respective transcription factors in the nucleus [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Wein M.N.
      • et al.
      Salt-inducible kinases: physiology, regulation by cAMP, and therapeutic potential.
      ]. Both class IIa HDACs and CRTCs have relevant roles in metabolic homeostasis, inflammation, and tumorigenesis and alterations in these proteins have been reported in lung pathologies.
      The role of SIKs in remodeling processes is further achieved through the regulation of TGF-β1 [
      • Hutchinson L.D.
      • et al.
      Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses.
      ,
      • Bertorello A.M.
      • et al.
      Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).
      ], phosphatidylinositol 3-kinase (PI3K)–AKT signaling [
      • Cai Y.
      • et al.
      Salt-inducible kinase 3 promotes vascular smooth muscle cell proliferation and arterial restenosis by regulating AKT and PKA–CREB signaling.
      ,
      • Zhao J.
      • et al.
      SIK2 enhances synthesis of fatty acid and cholesterol in ovarian cancer cells and tumor growth through PI3K/Akt signaling pathway.
      ], and the Hippo–Yes-associated protein (Hippo–YAP) pathway [
      • Chen F.
      • et al.
      Salt-inducible kinase 2: an oncogenic signal transmitter and potential target for cancer therapy.
      ,
      • Rong X.
      • et al.
      Molecular mechanisms of tyrosine kinase inhibitor resistance induced by membranous/cytoplasmic/nuclear translocation of epidermal growth factor receptor.
      ], and some findings suggest that different SIK members may play different regulatory roles. The PI3K–AKT–mTOR and Hippo–YAP pathways may also be linked via SIK2, with mutual activation between the two, but this crosstalk has not been clarified [
      • Chen F.
      • et al.
      Salt-inducible kinase 2: an oncogenic signal transmitter and potential target for cancer therapy.
      ].
      Interestingly, SIKs have also been implicated in the control of inflammatory pathways and are recognized as promising therapeutic targets for the modulation of cytokine responses on immune activation. Pharmacological and genetic pan-SIK inhibition decreases CRTC3 phosphorylation with subsequent increases in CREB-dependent expression of the anti-inflammatory cytokine IL-10, while simultaneously decreasing the expression of proinflammatory cytokines; namely, IL-1, IL-6, IL-12, and TNF-α [
      • Sundberg T.B.
      • et al.
      Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells.
      ,
      • Clark K.
      • et al.
      Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages.
      ]. Further research showed that protein kinase A (PKA)-dependent inhibition of SIKs favors HDAC4 dephosphorylation, which inhibits the activity of nuclear factor kappa B (NF-κB), a central mediator of immune and inflammatory responses, thus suppressing proinflammatory cytokine production by macrophages. Taken together, evidence demonstrates that pan-SIK inhibition has a robust anti-inflammatory effect, but some studies suggest that different SIK isoforms may play distinct roles. For example, a study showed that SIK2 and SIK3 are the major contributors to macrophage polarization and that inhibition of these isoforms resulted in increased IL-10 production and decreased IL-6, IL-12, and TNF-α production [
      • Darling N.J.
      • et al.
      Salt-inducible kinases are required for the IL-33-dependent secretion of cytokines and chemokines in mast cells.
      ,
      • Darling N.J.
      • et al.
      Inhibition of SIK2 and SIK3 during differentiation enhances the anti-inflammatory phenotype of macrophages.
      ]. Conversely, another study reported that SIK1 and SIK3 overexpression, but not SIK2, inhibited the activity of NF-κB and suppressed downstream proinflammatory cytokines [
      • Yong Kim S.
      • et al.
      Salt-inducible kinases 1 and 3 negatively regulate Toll-like receptor 4-mediated signal.
      ]. These findings may reflect distinct isoform roles but may also be the result of differential expression levels in distinct tissues, which deserves further investigation.
      Finally, SIKs are also recognized for their ability to regulate vascular tone, mainly through the modulation of Na+,K+-ATPase (NKA). In response to increases in intracellular sodium [
      • Sjöström M.
      • et al.
      SIK1 is part of a cell sodium-sensing network that regulates active sodium transport through a calcium-dependent process.
      ] or G protein-coupled receptor (GPCR) signals [
      • Jaitovich A.
      • Bertorello A.M.
      Intracellular sodium sensing: SIK1 network, hormone action and high blood pressure.
      ], SIK1 phosphorylates phosphatase methylesterase-1 (PME-1), causing its dissociation from the PP2A/PME-1/NKA complex. Consequently, PP2A then dephosphorylates the NKA α-subunit, which translates into increased NKA catalytic activity [
      • Sjöström M.
      • et al.
      SIK1 is part of a cell sodium-sensing network that regulates active sodium transport through a calcium-dependent process.
      ,
      • Popov S.
      • et al.
      Lack of salt-inducible kinase 2 (SIK2) prevents the development of cardiac hypertrophy in response to chronic high-salt intake.
      ]. Moreover, SIKs are also able to modulate NKA expression by simultaneously preventing CRTC entry into the nucleus and promoting ATP1B1 gene transcription, and by repressing the hormones that incite NKA expression [
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ,
      • Taub M.
      • et al.
      Renal proximal tubule Na,K-ATPase is controlled by CREB-regulated transcriptional coactivators as well as salt-inducible kinase 1.
      ]. Findings showing that lack of SIK1 in vascular SMCs translates into decreased NKA activity, which is associated with increased vascular tone [
      • Bertorello A.M.
      • et al.
      Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).
      ], support the role of SIK1 in the regulation of vasoconstriction mechanisms.

      SIKs at the crossroads of several regulatory mechanisms of PAH pathogenesis

      SIK substrates with established roles in PAH

      SIK regulatory functions in vasoconstriction, remodeling, and inflammation predict their potential therapeutic role in PAH and this is further supported by the fact that many SIK upstream regulators and downstream substrates are also involved in PAH pathogenesis (Figure 2, Key figure ).
      Figure 2
      Figure 2Key figure. Pathways regulated by salt-inducible kinases (SIKs) and shared by pulmonary arterial hypertension (PAH), cancer, inflammatory diseases, and hypertension.
      PAH is sustained by three major pathogenic mechanisms; vasoconstriction, inflammation, and remodeling. The role of SIKs in PAH is strongly suggested from their well-known functions in diseases that share the same pathological hallmarks. Since these kinases regulate a vast number of ion channels, growth factors, inflammatory molecules, and transcriptional factors that underlie these mechanisms, it is anticipated that SIKs may be important therapeutic targets with effects on multiple pathological levels. Accumulating evidence provides further support that the SIK1, SIK2, and SIK3 isoforms may have distinct regulatory functions. Abbreviations: HDAC, histone deacetylase; NKA, Na+,K+-ATPase; PI3K, phosphatidylinositol 3-kinase; TNF-α, tumor necrosis factor alpha; YAP, Yes-associated protein.
      HDACs are emerging as putative therapeutic targets in PAH, since histone acetylation is a critical epigenetic modification that strongly correlates with proliferative, apoptosis-resistant, inflammatory, and fibrotic vascular cell phenotypes. Both protein and mRNA expression of the class IIa HDACs 4, 5, and 7 are increased in the pulmonary arteries of a PAH experimental model and in patients’ lungs. Nuclear accumulation of HDACs mediates the decrease in myocyte enhancer factor 2 (MEF2) activity, essential for the maintenance of pulmonary vascular homeostasis [
      • Chen F.
      • et al.
      Inhibition of histone deacetylase reduces transcription of NADPH oxidases and ROS production and ameliorates pulmonary arterial hypertension.
      ,
      • Saygin D.
      • et al.
      Transcriptional profiling of lung cell populations in idiopathic pulmonary arterial hypertension.
      ]. Several pharmacological inhibitors of class IIa HDACs were found to restore MEF2 activity, decrease cell proliferation and migration, and mitigate experimental PAH, supporting the therapeutic potential of HDAC inhibition [
      • Kim J.
      • et al.
      Restoration of impaired endothelial myocyte enhancer factor 2 function rescues pulmonary arterial hypertension.
      ,
      • Chelladurai P.
      • et al.
      Targeting histone acetylation in pulmonary hypertension and right ventricular hypertrophy.
      ]. Because SIKs are suppressors of class IIa HDACs and are known to rescue MEF2-dependent transcription [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ], the upregulation of SIKs in the context of PAH opens a new direction in the discovery of a new disease-modifying strategy.
      The PI3K–AKT signaling pathway is now emerging as a central target in PAH, as strong evidence has established PI3K as a key regulator of pulmonary arterial SMC proliferation, migration, and survival. Both genetic and pharmacological inhibition of PI3K was able to prevent the onset and progression of and even reverse vascular remodeling as well as RV hypertrophy in three PAH animal models [
      • Berghausen E.M.
      • et al.
      Disrupted PI3K subunit p110α signaling protects against pulmonary hypertension and reverses established disease in rodents.
      ]. Recently, SIK2 and SIK3 isoforms were found to phosphorylate a regulatory subunit of the PI3K complex, followed by AKT phosphorylation and activation [
      • Cai Y.
      • et al.
      Salt-inducible kinase 3 promotes vascular smooth muscle cell proliferation and arterial restenosis by regulating AKT and PKA–CREB signaling.
      ,
      • Zhao J.
      • et al.
      SIK2 enhances synthesis of fatty acid and cholesterol in ovarian cancer cells and tumor growth through PI3K/Akt signaling pathway.
      ]. One study found that SIK3 regulates the proliferation and migration of aortic SMCs through mechanisms that involve CREB and AKT signaling. Inhibition of SIKs was able to decrease arterial SMC proliferation in vitro and suppress neointima formation in a rodent model of arterial injury [
      • Cai Y.
      • et al.
      Salt-inducible kinase 3 promotes vascular smooth muscle cell proliferation and arterial restenosis by regulating AKT and PKA–CREB signaling.
      ]. These findings suggest the potential therapeutic value of SIK2 and SIK3 inhibition in PAH.
      Hippo–YAP is another key pathway implicated in PAH pathophysiology that is simultaneously a substrate for SIKs. In PAH, YAP nuclear localization is increased, from where it promotes the proliferation and migration of SMCs [
      • He J.
      • et al.
      The role of Hippo/Yes-associated protein signalling in vascular remodelling associated with cardiovascular disease.
      ]. SIK2 promotes the expression of the proto-oncogene YAP through regulation of the Hippo–YAP pathway, suggesting that SIK2 inhibition may have therapeutic value in the context of PAH [
      • Chen F.
      • et al.
      Salt-inducible kinase 2: an oncogenic signal transmitter and potential target for cancer therapy.
      ,
      • Rong X.
      • et al.
      Molecular mechanisms of tyrosine kinase inhibitor resistance induced by membranous/cytoplasmic/nuclear translocation of epidermal growth factor receptor.
      ].
      Importantly, abnormal proliferation in PAH is also driven by enhanced TGF-β signaling. In vitro and in vivo studies have shown that SIK1 acts as a negative regulator of TGF-β [
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ,
      • Bertorello A.M.
      • et al.
      Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).
      ,
      • Kowanetz M.
      • et al.
      TGFβ induces SIK to negatively regulate type I receptor kinase signaling.
      ]. Specifically, ablation of SIK1 in mice resulted in a vascular increase of TGF-β expression in the context of systemic hypertension [
      • Bertorello A.M.
      • et al.
      Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).
      ]. Recently, pharmacological SIK inhibition was reported to suppress TGF-β-mediated transcription through a mechanism largely driven by the SIK2 and SIK3 isoforms [
      • Hutchinson L.D.
      • et al.
      Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses.
      ]. Together, these findings indicate that SIK1 overexpression and/or SIK2/3 inhibition should be further investigated as potential strategies to address PAH-associated remodeling.
      Moreover, numerous cytokines and chemokines that comprise the PAH inflammatory environment are also SIK substrates. In PAH, the pulmonary vasculature is marked by aberrant secretion of IL-1, IL-6, and TNF-α [
      • Hu Y.
      • et al.
      Perivascular inflammation in pulmonary arterial hypertension.
      ]. Pan-SIK inhibition, both in vitro and in vivo, leads to decreased expression of these cytokines, and this mechanism seems to be mostly mediated by the SIK2 isoform [
      • Sundberg T.B.
      • et al.
      Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells.
      ,
      • Clark K.
      • et al.
      Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages.
      ,
      • Darling N.J.
      • et al.
      Inhibition of SIK2 and SIK3 during differentiation enhances the anti-inflammatory phenotype of macrophages.
      ,
      • Sundberg T.B.
      • et al.
      Development of chemical probes for investigation of salt-inducible kinase function in vivo.
      ]. By contrast, SIK1 overexpression was shown to attenuate TNF-α and IL-6 expression in activated macrophages [
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ,
      • Yong Kim S.
      • et al.
      Salt-inducible kinases 1 and 3 negatively regulate Toll-like receptor 4-mediated signal.
      ]. Although the precise role of SIK3 in inflammation remains controversial, accumulating evidence suggests that selective SIK2 inhibition or SIK1 overexpression could represent a new therapeutic approach to PAH-associated inflammation.

      Lessons learned from AMPK in PAH

      SIKs integrate a family of 14 AMPK-related kinases that rely on phosphorylation by liver kinase B1 (LKB1) for their catalytic activity (see Figure I in Box 1). Both AMPK, the representative kinase of the family, and SIKs contain an activation loop in their kinase domains and share a high degree of sequence similarity within the activation loop and the gatekeeper [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ,
      • Sakamoto K.
      • et al.
      The salt-inducible kinases: emerging metabolic regulators.
      ]. However, unlike AMPK, SIKs are constitutively T-loop phosphorylated and so their intrinsic activity is generally regulated by multiple signals through complex LKB1-independent mechanisms, while AMPK is primarily activated by cellular energy levels (Box 1) [
      • Sakamoto K.
      • et al.
      The salt-inducible kinases: emerging metabolic regulators.
      ].
      AMPK and SIKs also recognize common phosphorylation motifs and thus share a variety of downstream substrates, like CRTC2 and HDAC4, which suggests that some physiological and pathological functions of the two kinases may overlap [
      • Sakamoto K.
      • et al.
      The salt-inducible kinases: emerging metabolic regulators.
      ].
      Endothelial AMPK is downregulated in PAH animal models and patients with PAH, and EC-specific deletion of AMPK accelerated and worsened hypoxia-induced PAH [
      • Zhao Q.
      • et al.
      AMPK and pulmonary hypertension: crossroads between vasoconstriction and vascular remodeling.
      ]. By contrast, studies in pulmonary vascular SMCs reported contradictory results. A significant amount of evidence found that AMPK is decreased in SMCs from PAH patients and hypoxia-induced mice compared with respective controls. A few studies, however, reported that AMPK was upregulated, although these inconsistencies may be due to differences in experimental procedures such as the isolation of more proximal artery segments of larger diameter, less affected by PAH and where the AMPK-α1 subunit is less active [
      • Zhao Q.
      • et al.
      AMPK and pulmonary hypertension: crossroads between vasoconstriction and vascular remodeling.
      ].
      Mechanistically, AMPK in ECs plays a preventive role in PAH, promoting an increase in nitric oxide (NO) production and concurrently decreasing endothelin-1 (EN-1) secretion. Endothelial AMPK is also responsible for stabilizing angiotensin-converting enzyme 2 (ACE2), which increases the expression of the vasodilator angiotensin 1–7, and for mediating calcium influx in microvascular ECs [
      • Shen H.
      • et al.
      MDM2-mediated ubiquitination of angiotensin-converting enzyme 2 contributes to the development of pulmonary arterial hypertension.
      ].
      By contrast, in pulmonary vascular SMCs, AMPK participates in molecular pathways that presumably increase intracellular calcium levels and promote vasoconstriction, inhibiting voltage-dependent potassium channels, activating voltage-gated calcium channels, and inducing calcium release from the sarcoplasmic reticulum [
      • Zhao Q.
      • et al.
      AMPK and pulmonary hypertension: crossroads between vasoconstriction and vascular remodeling.
      ]. Nonetheless, most studies report that AMPK inhibition promotes the proliferation and survival of pulmonary SMCs. Pharmacological activation of AMPK by metformin or AICAR improves PAH in four different experimental models, inhibits SMCs proliferation, and promotes survival [
      • Zhao Q.
      • et al.
      AMPK and pulmonary hypertension: crossroads between vasoconstriction and vascular remodeling.
      ,
      • Yoshida T.
      • et al.
      Metformin prevents the development of monocrotaline-induced pulmonary hypertension by decreasing serum levels of big endothelin-1.
      ,
      • Liu Y.
      • et al.
      Metformin prevents progression of experimental pulmonary hypertension via inhibition of autophagy and activation of adenosine monophosphate-activated protein kinase.
      ,
      • Huang X.
      • et al.
      Regulatory effect of AMP-activated protein kinase on pulmonary hypertension induced by chronic hypoxia in rats: in vivo and in vitro studies.
      ,
      • Liao S.
      • et al.
      Chronic dosing with metformin plus bosentan decreases in vitro pulmonary artery contraction from isolated arteries in adults with pulmonary hypertension.
      ]. Although the detailed mechanisms underlying the benefits of AMPK activation remain elusive, recent findings suggest that AMPK inhibits myosin light chain (MLC) kinases and MLC phosphorylation, which suppresses vascular SMC contraction. Furthermore, autophagy associated with hypoxia-induced PAH is reversed by AMPK activation with metformin [
      • Liu Y.
      • et al.
      Metformin prevents progression of experimental pulmonary hypertension via inhibition of autophagy and activation of adenosine monophosphate-activated protein kinase.
      ].
      The benefits of AMPK activation are not limited to the lung and several studies have demonstrated that metformin and other AMPK activators reduce RV systolic pressure and remodeling, all contributing to improved hemodynamics [
      • Yoshida T.
      • et al.
      Metformin prevents the development of monocrotaline-induced pulmonary hypertension by decreasing serum levels of big endothelin-1.
      ,
      • Liu Y.
      • et al.
      Metformin prevents progression of experimental pulmonary hypertension via inhibition of autophagy and activation of adenosine monophosphate-activated protein kinase.
      ,
      • Huang X.
      • et al.
      Regulatory effect of AMP-activated protein kinase on pulmonary hypertension induced by chronic hypoxia in rats: in vivo and in vitro studies.
      ]. This is in line with findings of reduced AMPK activity during the late stage of heart failure [
      • Deng Y.
      • et al.
      Altered mTOR and Beclin-1 mediated autophagic activation during right ventricular remodeling in monocrotaline-induced pulmonary hypertension.
      ].
      Metformin is currently under evaluation for PAH treatment in two Phase 2 clinical trials (Clinical Trial Numbersi: NCT03617458 and NCT03629340) and one observational study (Clinical Trial Numberi: NCT01884051). Results to date in a small population reported an improvement in RV function in PAH patients under metformin therapy, although the 6-min walk test did not change. However, previously Liao et al. [
      • Liao S.
      • et al.
      Chronic dosing with metformin plus bosentan decreases in vitro pulmonary artery contraction from isolated arteries in adults with pulmonary hypertension.
      ] reported that metformin added to the classic therapy with bosentan significantly improved the 6-min walk test and RV hemodynamic parameters and decreased pulmonary EN-1 compared with monotherapy. These preliminary results reveal potential benefits of AMPK activation in the context of PAH.
      Interestingly, other cardiovascular agents were also found to ameliorate experimental PAH via AMPK activation [
      • Prisco S.Z.
      • et al.
      With no lysine kinase 1 promotes metabolic derangements and RV dysfunction in pulmonary arterial hypertension.
      ]. A novel drug in development successfully prevented hypoxia-induced PAH with the activation of endothelial NO synthase (eNOS) and inhibition of ERK1/2 via AMPK phosphorylation [
      • Jin Q.
      • et al.
      C1q/TNF-related protein-9 ameliorates hypoxia-induced pulmonary hypertension by regulating secretion of endothelin-1 and nitric oxide mediated by AMPK in rats.
      ]. Ras/Raf/ERK signaling is downregulated by both AMPK and SIKs, which is of relevance since this is a proliferative pathway upregulated in PAH [
      • Kurakula K.
      • et al.
      Endothelial dysfunction in pulmonary hypertension: cause or consequence?.
      ,
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ,
      • Jin Q.
      • et al.
      C1q/TNF-related protein-9 ameliorates hypoxia-induced pulmonary hypertension by regulating secretion of endothelin-1 and nitric oxide mediated by AMPK in rats.
      ].
      AMPK and SIKs also share other substrates relevant in PAH pathophysiology, such as CRTCs and class IIa HDACs. Both kinases are able to phosphorylate HDAC4 and HDAC5, promoting their retention in the cytosol, thus blocking their transcriptional regulatory functions in the nucleus [
      • Di Giorgio E.
      • Brancolini C.
      Regulation of class IIa HDAC activities: it is not only matter of subcellular localization.
      ].
      In summary, a significant body of evidence supports the histological and hemodynamic benefits of AMPK activation in PAH, establishing this kinase as a key player in pulmonary vasoconstriction and remodeling as well as in right-heart failure, which may suggest a role for SIKs in PAH. Considering that the two kinases share similar structures, common upstream regulators, and common downstream substrates, it is anticipated that SIKs may have similar pathological functions. Thus, further research will be needed to elucidate the relative contributions of AMPK and SIKs to the development and progression of PAH.

      Lessons learned from hypertension

      Elevated arterial blood pressure is a consequence of both the increased contractility of SMCs and the abnormal vascular remodeling with extracellular matrix deposition. SIK1 regulates contractility mainly through NKA modulation [
      • Popov S.
      • et al.
      Salt-inducible kinase 1 influences Na+,K+-ATPase activity in vascular smooth muscle cells and associates with variations in blood pressure.
      ,
      • António T.
      • et al.
      The role of salt-inducible kinases on the modulation of renal and intestinal Na+,K+-ATPase activity during short- and long-term high-salt intake.
      ]. In vascular SMCs, SIK1 correlates with increased NKA activity, lower blood pressure, and downregulation of the TGF-β1 pathway [
      • Bertorello A.M.
      • et al.
      Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).
      ]. One human genotype–phenotype association study including population-based cohorts found that a human polymorphism causing higher SIK1 activity triggered the increase of NKA activity, leading to lower systolic and diastolic blood pressures [
      • Popov S.
      • et al.
      Salt-inducible kinase 1 influences Na+,K+-ATPase activity in vascular smooth muscle cells and associates with variations in blood pressure.
      ].
      Importantly, key players in pulmonary vasoconstriction like EN-1, the EN-1 receptor, and EN-converting enzyme were upregulated in cultured vascular SMCs and aortas from SIK1-null mice. In vitro experiments showed that loss of SIK1 was required for SMCs to acquire a contractile phenotype through transcriptional upregulation of contractile genes and EN-1 in the presence of TGF-β1 [
      • Bertorello A.M.
      • et al.
      Increased arterial blood pressure and vascular remodeling in mice lacking salt-inducible kinase 1 (SIK1).
      ].
      Although pulmonary vessels are distinct from systemic vessels, both comprise heterogeneous populations of SMCs that share similar regulatory mechanisms. Proteomic studies revealed that only 19 of 198 proteins were differently expressed between human pulmonary arterial SMCs and human aortic SMCs, suggesting that proteins with regulatory roles in systemic hypertension, such as SIK1, should play similar functions in PAH [
      • Régent A.
      • et al.
      Proteomic analysis of vascular smooth muscle cells in physiological condition and in pulmonary arterial hypertension: toward contractile versus synthetic phenotypes.
      ]. Interestingly, a mutation on the ATP1A2 gene, encoding the α2-subunit of NKA, was reported in a case of hereditary PAH, further reinforcing the hypothesis of a pathophysiological link [
      • Montani D.
      • et al.
      Pulmonary arterial hypertension in familial hemiplegic migraine with ATP1A2 channelopathy.
      ].

      Lessons learned from cancer

      It has become increasingly clear that PAH can be considered a cancer-like disease, as research reveals a broad number of pathogenic mechanisms in common with cancer (Figure 2). PAH and malignant cells share a hyperproliferative and antiapoptotic profile with a metabolic shift towards glycolysis, but not the tissue invasion and metastasis hallmarks. Other features of PAH such as persistent inflammation, gene mutations and immune evasion also resemble cancer hallmarks [
      • Dabral S.
      • et al.
      A RASSF1A–HIF1α loop drives Warburg effect in cancer and pulmonary hypertension.
      ].
      Findings of elevated pulmonary pressure and extensive vascular remodeling in a great number of patients with lung cancer further supports the analogy between cancer and PAH [
      • Cool C.D.
      • et al.
      The hallmarks of severe pulmonary arterial hypertension: the cancer hypothesis-ten years later.
      ,
      • Pullamsetti S.S.
      • et al.
      Cancer and pulmonary hypertension: learning lessons and real-life interplay.
      ].
      Although nonmetastatic, PAH has a cancer-like nature that provides an opportunity to predict unknown pathogenic mechanisms and exploit cancer therapeutic targets to treat PAH.
      LKB1 is a well-recognized tumor suppressor and a large number of mutations in its gene are related to several forms of cancer, including lung cancer. LKB1 is known to activate AMPK-related kinases, but which member mediates its tumor-suppressive activity remains elusive. AMPK, the best studied of these LKB1-dependent kinases, was hypothesized to be responsible for this oncogenic suppression, but soon several studies revealed that AMPK is required for cancer cell survival, and some even found that AMPK deletion inhibited tumorigenesis. A recent study proposed that SIKs could mediate LKB1 antitumorigenic effects and discovered that SIK1 and SIK3 were mediators of the tumor-suppression effects of LKB1 in non-small lung cancer [
      • Hollstein P.E.
      • et al.
      The AMPK-related kinases SIK1 and SIK3 mediate key tumor-suppressive effects of LKB1 in NSCLC.
      ].
      SIKs have attracted interest in the cancer field and were found to be important players in ovarian, breast, lung, and many other forms of cancer. SIKs modulate several signaling pathways relevant in oncogenesis that are also known to be part of PAH pathogenesis.
      SIK2 is the best-characterized isoform in cancer and consistent findings suggest it promotes tumorigenesis, increases EndMT facilitating malignant cell migration, and promotes glycolytic metabolism via the PI3K/AKT/HIF1α signaling pathway. SIK2 is overexpressed in several forms of cancer, including ovarian and prostate cancers, where its suppression resulted in cell-cycle arrest and mitigated metastasis [
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ].
      Conversely, SIK1 and SIK3 were reported to function as potent tumor suppressors [
      • Murray C.W.
      • et al.
      An LKB1–SIK axis suppresses lung tumor growth and controls differentiation.
      ]. The antitumorigenic effects of SIK1 have been reported in malignancies such as breast, ovarian, colorectal and lung cancers and was associated with a decrease in cell proliferation, epithelial-to-mesenchymal transition, and in the IL-6/JAK/STAT pathway. Since these processes are upregulated not only in cancer but also in PAH, it is anticipated that SIK1 may also display these beneficial effects in PAH [
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ].
      The role of SIK3 in cancer is understudied and findings are contradictory. Some authors suggest that SIK3 promotes proliferation, cell growth, and cell-cycle progression, thus acting as an oncogene, while others found that low SIK3 expression was linked to poor survival [
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Zhang X.-W.
      • et al.
      Salt-inducible kinase inhibition sensitizes human acute myeloid leukemia cells to all-trans retinoic acid-induced differentiation.
      ,
      • Tarumoto Y.
      • et al.
      Salt-inducible kinase inhibition suppresses acute myeloid leukemia progression in vivo.
      ]. Therefore, it is not possible to predict an analogous role in PAH.

      Pharmacological modulation of SIKs

      The development of small-molecule inhibitors for dysregulated kinases has been a major focus of oncological and inflammatory research. SIK inhibition might be valuable in the context of pathologies like osteoporosis, cancer, and possibly metabolic and inflammatory diseases. Several SIK inhibitors are currently available for research purposes, while others have advanced to clinical trials (Table 1).
      Table 1Most relevant SIK modulators available to date
      Abbreviations: EMA, European Medicines Agency; NCT, National Clinical Trial numberi; n/a, not available; +, low selectivity; ++, medium selectivity; +++, high selectivity.
      Relative selectivity between SIK isoformsAdditional targetPreclinical findingProspective therapeutic useDevelopment phaseRefs
      SIK1SIK2SIK3
      Staurosporine++++n/aBroad kinase inhibitor[
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ]
      Diosmetinn/a++n/aBroad inhibitor[
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ]
      Dasatinib+++++Tyrosine kinases• Inhibits macrophage polarization

      • Promotes TGF-β-mediated apoptosis
      FDA and EMA approved for chronic myeloid leukemia and lymphoblastic leukemia[
      • Ozanne J.
      • et al.
      The clinically approved drugs dasatinib and bosutinib induce anti-inflammatory macrophages by inhibiting the salt-inducible kinases.
      ]
      Bosutinib+++++Tyrosine kinases• Inhibits macrophage polarization

      • Promotes TGF-β-mediated apoptosis
      Intracerebral hemorrhage-associated inflammationFDA and EMA approved for chronic myeloid leukemia[
      • Ozanne J.
      • et al.
      The clinically approved drugs dasatinib and bosutinib induce anti-inflammatory macrophages by inhibiting the salt-inducible kinases.
      ]
      Crenolanibn/a+++Tyrosine kinasesAcute myeloid leukemiaSeveral completed clinical trials, but not yet approved[
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ]
      HG-9-91-01++++Src family kinases and other tyrosine and serine/threonine kinases• Attenuates colitis in mouse model

      • Promotes pancreatic β cells proliferation
      For in vitro studies only[
      • Sakamoto K.
      • et al.
      The salt-inducible kinases: emerging metabolic regulators.
      ]
      YKL-05-099++++++Tyrosine kinases• Inhibits macrophage polarization

      • Increased IL-10, decreased IL-6, IL-12, and TNF-α in vivo
      For in vitro and in vivo studies only[
      • Sundberg T.B.
      • et al.
      Development of chemical probes for investigation of salt-inducible kinase function in vivo.
      ]
      YKL-06-061++++++Tyrosine kinasesFor in vitro and in vivo studies only[
      • Darling N.J.
      • Cohen P.
      Nuts and bolts of the salt-inducible kinases (SIKs).
      ,
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ]
      MRT-199665++++++Other AMPK-related kinases• Increased IL-10, decreased IL-6, IL-12, and TNF-α in vivoFor in vitro and in vivo studies only[
      • Clark K.
      • et al.
      Phosphorylation of CRTC3 by the salt-inducible kinases controls the interconversion of classically activated and regulatory macrophages.
      ]
      ARN-3236++++++JAK2, LCK, NUAK2, SRPK1, VEGFR2• Enhances sensitivity of ovarian cancer xenograft models to carboplatin and paclitaxelBreast and ovarian cancerPreclinical[
      • Sakamoto K.
      • et al.
      The salt-inducible kinases: emerging metabolic regulators.
      ,
      • Fan D.
      • et al.
      A novel salt inducible kinase 2 inhibitor, ARN-3261, sensitizes ovarian cancer cell lines and xenografts to carboplatin.
      ]
      ARN-3261++++++n/a• Enhances sensitivity of ovarian cancer xenograft models to carboplatin and paclitaxelBreast and ovarian cancerPhase 1 ongoing for ovarian cancerNCT04711161
      MRIA9+++++++Only PAK• Enhances sensitivity of ovarian cancer 3D spheroids to paclitaxelOvarian cancer[
      • Tesch R.
      • et al.
      Structure-based design of selective salt-inducible kinase inhibitors.
      ]
      Pterosin B+++n/aOsteoarthritis[
      • Sun Z.
      • et al.
      The potent roles of salt-inducible kinases (SIKs) in metabolic homeostasis and tumorigenesis.
      ]
      GLPG 4399++++++n/aInflammatory diseasesPhase 1 completedNCT04653467
      GLPG 3970++++++n/aInflammatory diseasesPhase 1 completed for psoriasis, Phase 2 ongoing for rheumatoid arthritis, Sjögren’s syndrome, ulcerative colitisNCT04106297

      NCT04577781

      NCT04700280

      NCT04577794
      GLPG 3312++++++n/aInflammatory diseasesPhase 1 completedNCT03800472
      a Abbreviations: EMA, European Medicines Agency; NCT, National Clinical Trial numberi; n/a, not available; +, low selectivity; ++, medium selectivity; +++, high selectivity.
      HG-9-91-01 is one of the most potent pan-SIK inhibitors available that does not inhibit other kinases from the AMPK family. This owes to the fact that it not only targets the ATP-binding site, but also binds SIKs’ unique hydrophobic pocket at the gatekeeper site. However, the pharmacokinetic properties of HG-9-91-01 made it incompatible with in vivo studies, while this problem was overcome by its analog YKL-05-99. Mechanistically, these inhibitors promote dephosphorylation and nuclear translocation of CRTCs and HDACs, enabling their function as transcriptional regulators. In pancreatic β cells, for example, this translates into enhanced proliferation through activation of the mTOR and ATF6 signaling pathways.
      Research efforts from several academic groups to optimize SIK modulators led to the development of small molecules such as ARN-3236, the first orally administered, with improved selectivity towards SIK2 that successfully enhanced ovarian cancer cell chemosensitivity to paclitaxel in a xenograft model [
      • Zhou J.
      • et al.
      A novel compound ARN-3236 inhibits salt-inducible kinase 2 and sensitizes ovarian cancer cell lines and xenografts to paclitaxel.
      ].
      Compounds HG-9-91-01, YKL-05-099, and ARN-3236 (Table 1) possess different selectivity profiles and potencies towards SIK isoforms, but all share the fact that they inhibit Src family protein tyrosine kinases and other protein kinases. MRIA9 was developed to circumvent this obstacle and, although slightly less potent, it inhibits only one off target while retaining the sensitizing effect on ovarian cancer cells to paclitaxel [
      • Tesch R.
      • et al.
      Structure-based design of selective salt-inducible kinase inhibitors.
      ].
      Recently, SIK inhibition has also gained the attention of the pharmaceutical industry, which has focused particularly on the development of SIK2 and SIK3 inhibitors. Galapagos NV has an ongoing program focused on the prophylaxis and treatment of inflammation and autoimmune diseases through SIK inhibition. GLPG 4399, GLPG 3970, and GLPG 3312 are the compounds in the most advanced development phases and are reported to have a dual mode of action, both increasing the transcription of anti-inflammatory cytokines and inhibiting the transcription of proinflammatory cytokines. Phase 1 clinical trials revealed that these inhibitors are well tolerated, and in 2021 GLPG 3970 progressed to Phase 2 clinical trials for potential therapeutic use in rheumatoid arthritis (Clinical Trial Numberi: NCT04577781), Sjögren’s syndrome (Clinical Trial Numberi: NCT04700280), and ulcerative colitis (Clinical Trial Numberi: NCT04577794). Inhibitors with improved selectivity towards SIK2 could be useful in the management of PAH.
      Conversely, evidence suggests that selective activation of SIK1 and SIK3 could constitute a multitarget approach, simultaneously decreasing vasoconstriction, remodeling, and inflammation. However, no SIK activators are presently available. While inhibition of protein kinases is relatively easy to achieve with the use of small molecules to disturb the catalytic kinase domain, enzyme activators are difficult to develop and require a specific approach to the kinase’s activation mechanism. This is because most enzymes do not possess activator-binding sites, while all contain an active site susceptible to inhibition. Understanding the structure and activation mechanism of a kinase is critical for drug discovery, but SIK crystal structures are still lacking, which further restricts the discovery of SIK activators through structure-based design strategies.
      Although presently there are no SIK inhibitors in clinical use, the FDA-approved antineoplastics dasatinib and bosutinib were identified as potent nonselective SIK inhibitors [
      • Hutchinson L.D.
      • et al.
      Salt-inducible kinases (SIKs) regulate TGFβ-mediated transcriptional and apoptotic responses.
      ,
      • Sakamoto K.
      • et al.
      The salt-inducible kinases: emerging metabolic regulators.
      ]. While dasatinib and bosutinib also target other protein kinases, their anti-inflammatory and antiproliferative effects were shown to be largely mediated by SIKs. In vitro studies found that both dasatinib and bosutinib reduce phosphorylated CRTC3, promoting CREB-dependent transcription – namely, that of IL-10 – and this effect was absent with a mutant drug-resistant SIK2 [
      • Ozanne J.
      • et al.
      The clinically approved drugs dasatinib and bosutinib induce anti-inflammatory macrophages by inhibiting the salt-inducible kinases.
      ,
      • Ma L.
      • et al.
      Bosutinib attenuates inflammation via inhibiting salt-inducible kinases in experimental model of intracerebral hemorrhage on mice.
      ]. Moreover, these small-molecule inhibitors also decreased TGF-β-mediated transcriptional and apoptotic responses through SIK inhibition. Interestingly, clinical reports established PAH as an important adverse event of dasatinib and studies reported that dasatinib-pretreated rats were predisposed to PAH development [
      • Boucherat O.
      • et al.
      The cancer theory of pulmonary arterial hypertension.
      ]. Together, these findings support the possibility that selective SIK2 inhibition may be well tolerated in vivo and may have therapeutic potential for PAH.
      A recent study provided direct evidence in establishing a role for SIKs in the context of PAH. Zou et al. [
      • Zou L.
      • et al.
      Salt-inducible kinase 2 (SIK2) inhibitor ARN-3236 attenuates bleomycin-induced pulmonary fibrosis in mice.
      ] found that the expression of SIK2 and downstream phosphorylated CRTC2 is increased in the lungs of bleomycin-induced mice, an experimental model of pulmonary hypertension with pulmonary fibrosis. More importantly, ARN-3236, a potent and selective SIK2 inhibitor, successfully reduced phosphorylated CRCT2 and inhibited TGF-β1 signaling. In addition, proliferative and profibrotic mechanisms were suppressed, which resulted in decreased pulmonary inflammation and fibrosis in the bleomycin-induced mice. Future studies addressing the role of SIK2 in bleomycin-induced mice should include the assessment of histological changes in pulmonary arteries, RV systolic pressure, and RV hypertrophy.

      Concluding remarks and future perspectives

      Therapeutic options for PAH are currently scarce and mainly address increased pulmonary pressure by promoting SMC relaxation, leaving many other pathological features unresolved, such as the persistent inflammatory environment and vascular remodeling, consequences of hyperproliferative, antiapoptotic, and EndMT mechanisms. It is now broadly accepted that curative therapeutic approaches for PAH should not only restore the balance between vasodilators and vasoconstrictors, but also exhibit antiproliferative and anti-inflammatory effects. In this Opinion, we have gathered relevant recent findings demonstrating the putative role that SIKs may play in PAH pathophysiology by regulating many signaling pathways underlying these pathological processes. Available evidence on the mechanistic functions of SIKs argues that these roles should translate to models that recapitulate PAH. These points justify further research into the development of pharmacological SIK modulators for PAH treatment, but multiple obstacles remain ahead (see Outstanding questions). Finally, the suboptimal management of PAH emphasizes the need for deeper molecular insights on the disease pathogenesis that will be critical for the identification and validation of novel therapeutic targets for this high-burden disease with unmet medical needs.
      Could SIKs play a role in the aberrant signaling pathways of PAH, similar to what is found in cancer, hypertension, or inflammatory disorders?
      Do SIKs act independently or synergistically on PAH? If synergistically, how do they crosstalk to have an impact on cells and tissues?
      Does SIK2 play a proliferative antiapoptotic role in PAH as in cancer? Do SIK1 and SIK3 have opposite functions as in cancer?
      Would activation/inhibition of SIKs in PAH be able to generate effects similar to those observed in cancer?
      What would be the impact of SIK ablation in experimental models of PAH?
      Will molecules able to interfere simultaneously with multiple SIK isoforms be more effective in treating PAH than single-target agents? What would be the long-term safety profile of such pharmacological agents?
      Do SIKs regulate additional substrates that can explain why SIKs modulate such a wide range of transcriptional genes?

      Acknowledgments

      T.A. is supported by a PhD scholarship from FCT (UI/BD/150818/2020). We are grateful to Laetitia Gaspar for providing assistance in figure preparation.

      Declaration of interests

      No interests are declared.

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      Glossary

      AMP-activated protein kinase (AMPK)
      heterotrimeric enzyme that functions as a cellular metabolic regulator and energy sensor. On ATP depletion, it inactivates anabolic ATP-consuming pathways and activates catabolic ATP-generating processes. It is also activated by several hormones and cytokines. Inactivates a number of metabolic enzymes involved in ATP-consuming cellular events including fatty acid, cholesterol and protein synthesis, and activates ATP-generating processes.
      cAMP response element-binding protein (CREB)-regulated transcriptional coactivators (CRTCs)
      increase CREB activity, promoting cell proliferation, differentiation, and metabolic switch, and for that reason play a key role in tumor progression and resistance to chemotherapy.
      Class IIa histone deacetylases (HDACs)
      repressors of transcription factors like MEF2, regulating physiological and pathological mechanisms such as those related to bone formation, vascular calcification, or leukemia.
      Endothelial nitric oxide (NO) synthase (eNOS)
      enzyme responsible for most of the NO produced in the vasculature. The NO is crucial for the regulation of vascular tone and cellular proliferation and migration, as well as leukocyte adhesion and platelet aggregation. Endothelial dysfunction associated with PAH is characterized by impaired NO production by eNOS.
      Endothelial-to-mesenchymal transition (EndMT)
      mechanism by which ECs lose cell-to-cell contact and undergo transition towards a mesenchymal-like phenotype. This comes with loss of endothelial markers, like vascular endothelial (VE)-cadherin and PECAM-1, and the gain of myofibroblast markers such α-SMA and vimentin.
      Liver kinase B1 (LKB1)
      serine/threonine protein kinase that regulates cell metabolism, proliferation, and migration and is known to function as a tumor suppressor.
      Na+,K+-ATPase (NKA)
      transmembrane protein responsible for maintaining high cytosolic potassium concentration and low cytosolic sodium concentration. NKA is crucial to keep a negative resting membrane potential and provide a driving force for secondary ion transport, while it simultaneously works as a signal transducer in multiple pathways. Its expression and function are essential for the regulation of vasculature contractility. In pulmonary arteries, hypoxia inhibits NKA, leading to membrane depolarization and consequent opening of calcium channels, which culminates in vasoconstriction.
      Nuclear factor kappa B (NF-κB)
      a ubiquitous transcription factor that regulates the expression of genes involved in inflammation, innate immunity, stress responses, cell proliferation, and survival. Increased NF-κB activity promotes the expression of proinflammatory cytokines and chemokines, antiapoptotic factors, cell-cycle regulators, and adhesion molecules. NF-κB expression is increased in the lungs of PAH rodent models and activated in the pulmonary vessels of PAH patients. It is recognized as a key proinflammatory factor in the development of PAH.