In Search of the Second Hit in PAH

Since the discovery of mutations in bone morphogenetic protein receptor type II (BMPR2) gene in patients with pulmonary arterial hypertension (PAH), growing evidence from human genetics has supported a critical role for imbalanced signaling of the transforming growth factor-β (TGF-β) family, such that deficient BMP and maladaptive TGF-β signaling appear to contribute^1,2. BMPRII expression is not only reduced in PAH patients harboring mutations in BMPR2, but also in mutation-negative idiopathic PAH (IPAH)³, suggesting additional environmental or epigenetic factors may down-regulate this receptor. Several possible mechanisms have been proposed, including estrogen signaling⁴, interleukin-6-mediated inflammation⁵, TNFα-induced cleavage⁶ and other factors driving the ubiquitination and degradation of BMPRII⁷. The reduced penetrance of PAH-associated BMPR2 mutations is consistent with the need for additional factors such as vascular injury and/or inflammation for triggering disease⁸. In search of this missing link, Abdul-Salam and colleagues describe in the current issue a novel pathway that may integrate hypoxia and inflammation to regulate endothelial BMPRII expression, and the balance between BMP and TGF-β signaling⁹.

This team had previously described a proteomic analysis of lungs from PAH patients and control subjects for differentially expressed proteins¹⁰. A key finding was increased expression of CLIC4 in PAH tissues, localized predominantly to endothelial cells (ECs) in vascular lesions, with corresponding increases in CLIC4 observed in plasma and blood-derived ECs from IPAH patients, and in rat models of pulmonary hypertension (PH)¹⁰. Increased levels of CLIC4 altered barrier function, survival and angiogenic activity of ECs. CLIC4 modulated p65-mediated activation of NFκB, which in turn regulated hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor and endothelin-1. Consistent with a pathogenetic role, CLIC4 knockout mice developed less severe PH under hypoxia¹¹. Spiekerkoetter and colleagues showed previously that CLIC4 is regulated by BMPRII in pulmonary arterial smooth muscle cells (PASMCs) and modulates cell motility by altering alignment of myosin and actin filaments and the distribution and activation of small GTPases¹², suggesting CLIC4 exerts cell type-dependent effects in the pulmonary vasculature (Figure 1). Importantly, despite its name CLIC4 can only form poorly selective ion channels in artificial bilayers; instead, it may function as a scaffolding protein coupling the membrane to the cytoskeleton via protein interactions¹³. Taken together, these studies demonstrated potentially important cell-specific roles of CLIC4 in regulating inflammation and aberrant vascular function in PAH.

Figure: Effects of CLIC4 in PAEC and PASMC.

CLIC4 expression and Arf6 activity are both increased in PH. In PAEC, CLIC4 acts via Arf6 to reduce BMPRII expression and signaling by promoting lysosomal degradation and reducing recycling of the receptor protein. CLIC4 also increases TGF-β and NFκB signaling, resulting in increased proliferation, inflammation and permeability; reduced apoptosis; dysregulated angiogenesis and vascular remodeling⁹. In PASMC, BMPRII signaling maintains expression of CLIC4, responsible for activation of Rac1 and RhoA and cytoskeletal rearrangements required for hPASMC migration¹². Solid arrows denote regulatory effects, dotted arrows denote translocation. Blue text describes the effect of CLIC4 signaling on cell homeostasis. MHCIIA, myosin heavy chain IIA; ROS, reactive oxygen species.

In the current study, a proteomic analysis of human pulmonary arterial endothelial cells (hPAECs) revealed interactions of CLIC4 with proteins involved in endosomal trafficking, lysosomal function and inflammation, including GTPase activating proteins GIT1, GIT2 and clathrin. CLIC4 enhanced activity of ADP Ribosylation Factor 6 (Arf6) by interacting with GIT1/GIT2 to maintain Arf6 in the active form. Using Arf6siRNA and pharmacological inhibition with secinH3, Arf6 was shown to mediate known functions of CLIC4 including activation of NFκB, HIF-1α and tube formation (Figure). Importantly, CLIC4 negatively regulated BMPRII expression and BMP9-SMAD1/5 signaling, while enhancing TGF-β-SMAD3 signaling in hPAECs⁹. CLIC4, acting via Arf6, was found to increase clathrin-mediated internalization and lysosomal degradation, while reducing gyrating clathrin responsible for recycling endosomal cargo to cell surface, thereby reducing BMPRII expression in hPAECs⁹ and perturbing the balance between BMP and TGF-β signaling. Interestingly, Arf6 activity was increased in blood-derived EC from IPAH patients⁹, suggesting the relevance of this mechanism in human disease. The mechanistic contribution of these findings was tested in animal models of PH, in which elevated CLIC4 was observed in conjunction with activation of Arf6 and NFκB and reduced BMPRII in the lung. Inhibiting CLIC4 and Arf6 using CLIC4siRNA and secinH3 attenuated experimental PH, reduced CLIC4/Arf activation and restored pulmonary BMPRII expression⁹. Thus, in pursuing downstream effectors and pathways modulated by CLIC4, Arf6 was uncovered as an important mediator at the interface between CLIC4, BMP/TGF-β signaling and inflammatory signaling. These findings provide a potential explanation for the reduction of BMPRII expression in the pulmonary vasculature of IPAH patients, addressing an incomplete understanding of how BMP signaling deficiency may arise in acquired forms of PAH, while defining Arf6 and modification of endosomal trafficking as potentially druggable targets in PAH.

These important findings raise several questions for subsequent investigation. For example, what are the drivers of increased CLIC4 expression in PAH? The authors found increased pulmonary expression of CLIC4 and Arf6 activity three days following monocrotaline injection, at which point BMPRII expression was reduced but PH and right ventricular hypertrophy were not yet established⁹. Early upregulation of CLIC4 in this model could represent a secondary response to changes related to the disease state, since CLIC4 expression is regulated by hypoxia, reactive oxygen species, DNA damage and inflammation¹¹. On the other hand, the authors suggest that BMPRII deficiency may induce a state of heighted inflammatory and oxidative stress that could induce CLIC4 in vivo⁹; however, in their own previous work, silencing BMPRII expression in ECs did not alter CLIC4 protein levels¹¹. A prior study demonstrated that TGF-β promotes the expression of CLIC4 and Schnurri-2 and their translocation to the nucleus of keratinocytes, whereby nuclear CLIC4 associates with phosphorylated SMAD2 and SMAD3 to prevent their dephosphorylation and inactivation¹⁴. It is possible that this positive feedback loop could propagate TGF-β signaling in PAH, worsening the imbalance between BMP and TGF-β while BMPRII expression is suppressed by CLIC4. Understanding the specific drivers of CLIC4 expression in the pulmonary vasculature could reveal early events in the development of PAH or factors driving its progression.

The identification of CLIC4-interacting proteins provide new mechanistic insights into the impact of CLIC4, now confirmed via CLIC4siRNA in SUGEN/hypoxia-exposed mice, consistent with the attenuated hypoxia-induced PH in CLIC4 knockout mice¹¹. Intriguingly, the chloride channel function of CLIC4 may not be required for regulation of NFκB and BMPRII, demonstrated by lack of impact of chloride channel blocker IAA-94. Changes in plasma membrane anion permeability caused by CLIC4 overexpression and potential electrophysiological effects of this protein deserve further investigation. In support of its specificity for Arf6, CLIC4 did not affect the closely related Arf1, a Golgi-associated Arf protein. The roles of Golgi-associated Arf proteins could be further investigated in the context of dysfunctional protein trafficking in PAH. While the authors demonstrated a direct interaction between CLIC4 and GIT1, the precise mechanisms by which this complex modulates Arf6 activity are a subject for further study. It was emphasized that CLIC4 regulates the activity, not the expression levels of Arf6, paralleling observations that Arf6 activity and not expression are increased in PAH blood-derived ECs. The current in vivo studies used secinH3, a small molecule inhibitor that blocks Arf6 activation by inhibiting the GDP for GTP exchange mediated by Arf guanine nucleotide exchange factors¹⁵. While the impact of secinH3 supports the role of Arf6, future studies could test the contribution of Arf6 in vivo by gene targeting or ablation. While there are safety concerns associated with secinH3, which caused hepatic insulin resistance in mice¹⁵, the current study found no gross abnormalities in the liver and other organs with secinH3 treatment. An upcoming clinical trial of the Arf6 inhibitor NAV-5093 (Navigen Pharmaceuticals) in acute respiratory distress syndrome may provide further insight on the safety of inhibiting Arf6 (http://www.a6pharmaceuticals.com/technology) while providing a route for translation.

The authors suggest that CLIC4/Arf6-induced clathrin-mediated endocytosis, lysosomal trafficking and acidification are the principal drivers of reduced BMPRII expression in PAH⁹. Previous studies demonstrated that BMPRII expression in vascular cells is constitutively regulated via a lysosomal degradative pathway⁷ that can be blocked using lysosomal inhibitor chloroquine, and in this context the influence of CLIC4-regulated gyrating clathrin and ubiquitination on lysosomal trafficking could be clarified. Subsequent investigations could explore how CLIC4 enhances TGFβ-SMAD3 signaling, and why BMPRII appears to be preferentially targeted by CLIC4 signaling. Finally, the differential impact of CLIC4 on the endothelium versus pulmonary arterial smooth muscle¹² could be revisited in light of the current proposal to inhibit CLIC4/Arf6 systemically.

In summary, Abdul-Salam and colleagues have reported a novel mechanism by which CLIC4 regulates Arf6 in the endothelium to attenuate BMPRII-mediated BMP9 signaling and potentiate TGF-β, linking CLIC4 to the critical BMP/TGFβ signaling pathway. Activation of CLIC4 signaling by environmental stressors such as hypoxia and inflammation represent a plausible ‘second hit’ for promoting the imbalance of signaling in PAH and may provide a new therapeutic route for intervention.