Nuclear Focal Adhesion Kinase: Novel Endogenous Antagonist of Focal Adhesion Kinase-Mediated Smooth Muscle Proliferation and Neointima Formation

Atherosclerosis and restenosis are chronic and acute inflammatory vascular diseases, respectively, characterized by significant vascular remodeling^1,2. Pathological vascular remodeling in these settings is associated with activation and proliferation of resident vascular smooth muscle cells (SMCs) and recruitment and activation of immune cells, in particular macrophages³. For years, treatment of atherosclerosis has focused on intensive lipid lowering⁴ or surgical interventions, such as drug-eluting stent (DES) deployment. Current FDA-approved DES are based on paclitaxel or rapamycin derivatives, both general cell cycle inhibitors, and restenosis and stent thrombosis remain important complications of their use. Therefore, identification of newer generation therapeutic approaches would address a major unmet medical need. In addition, while lipid lowering therapy has been effective for many patients, many more patients remain at high risk for myocardial infarction or stoke due to a residual inflammatory risk rather than residual cholesterol risk⁵. Proof-of-principle for the treatment of underlying inflammation in these at-risk patients lies in the recent success of the CANTOS clinical trial⁶, which has opened up a new approach for cardiovascular disease prevention and treatment.

Differentiated SMCs are highly specialized cells that express high levels of contractile proteins necessary for their primary role of maintaining vessel homeostasis, vessel tone, blood pressure, and blood flow distribution. Under pathological conditions, however, SMCs are capable of undergoing profound phenotypic and functional changes resulting in a proliferative, inflammatory, pro-fibrotic phenotype⁷. As a result, mature SMCs play a critical role in pathological vascular remodeling. The concept of functional SMC phenotypic modulation is well accepted. However, while a complete understanding is critical to enable therapeutic advances in the treatment of vascular diseases, the mechanisms regulating SMC phenotypic transitions are complex. In contrast to the many individual and likely redundant pathological factors up-regulated in diseased vessels that contribute to disease progression, identification of nodal downstream molecules regulating SMC phenotype control is critical to identify novel therapeutics for the treatment or regression of atherosclerosis and restenosis.

There are multiple examples in cardiovascular research of “accidental” findings leading to seminal discoveries. Arguably one of the most famous in more recent years was the discovery of endothelial-derived nitric oxide and follow-up studies describing its function as a potent vasodilator. From the serendipitous discovery of warfarin to the accidental discovery of percutaneous transluminal angioplasty, the role of unexpected findings in guiding paradigm-shifting science has had an impact on the management of cardiovascular diseases. Such an unexpected finding was uncovered by Lim and colleagues when they identified an entirely novel and kinase-independent function for focal adhesion kinase (FAK)⁸. In endothelial cells, FAK is an important regulator of integrin-extracellular matrix (ECM) and inflammatory signaling. Subsequent findings by this group demonstrated that inhibition of FAK kinase activity inhibited TNFα-induced VCAM expression in endothelial cells⁹. Surprisingly, FAK kinase inhibition resulted in FAK nuclear localization, which promoted direct interaction with and FAK-dependent degradation of transcription factors required for TNFα-dependent VCAM production, thus uncovering an opposing and kinase-independent function for FAK in the regulation of inflammation⁹. In this issue of Circulation Research, using in vitro approaches and pharmacological approaches combined with a genetic SMC-specific FAK kinase dead mouse model, Jeong, et. al. now demonstrate that inhibition of FAK kinase activity promotes FAK nuclear localization that is associated with decreased SMC proliferation and injury-mediated neointima formation¹⁰. Mechanistically, this group demonstrates that nuclear FAK facilitates proteasome-mediated degradation of the transcription factor, GATA4, which contributes to loss of GATA4-induced cyclin D1 activity and decreased SMC proliferation. Thus, a novel endogenous inhibitory function for FAK in SMCs has been uncovered.

FAK is a non-receptor tyrosine-phosphorylated tyrosine kinase and kinase-independent scaffolding protein that localizes to integrin-rich focal adhesion sites¹¹. It is known to mediate integrin and growth factor signaling pathways to regulate SMC proliferation and motility. Importantly, SMC-specific depletion of FAK was shown to inhibit injury-induced neointima formation supporting its role in facilitating pathological vascular remodeling¹². FAK is a 125-kDa protein that consists of an N-terminal FERM domain that facilitates its protein scaffolding function, a central kinase domain, proline-rich regions, and a C-terminal focal adhesion targeting (FAT) domain that promotes localization of FAK to focal adhesions¹¹. Previous studies identified an endogenous inhibitor of FAK, FAK-related nonkinase (FRNK), as a SMC-specific regulator of FAK activity¹³. FRNK is expressed as an independent protein containing the C-terminal FAT domain, but lacking kinase activity. FRNK inhibits FAK activity by competitively interacting with FAK binding partners in focal adhesions or through recruitment of negative regulators of FAK to focal complexes. Thus, these earlier data revealed a novel, endogenous, and SMC-specific mechanism in place to regulate integrin- and growth factor-mediated FAK activation and SMC function. The current study highlights a new endogenous mechanism of FAK regulation, placing FAK as an essential regulator of pathological vascular remodeling and a potential novel therapeutic target for the treatment of vascular disease.

Transcription factors of the GATA family bind the consensus DNA sequence (A/T)GATA(A/G) through their highly conserved zinc finger domain(s), and in a tissue specific manner control cell proliferation and migration in development and diseases. Of all GATA family members, GATA4 and GATA6 are highly expressed in the vasculature during early embryonic development. While GATA4 expression is dampened in postnatal SMCs, GATA6 plays an important role in SMC phenotype maintenance. GATA6 is suppressed in vascular injury and, in contrast GATA6 overexpression promotes cell cycle arrest and a contractile, quiescent SMC phenotype and rescues injury-induced SMC contractile gene repression and neointima formation¹⁴. Moreover, GATA6 exerts distinctive roles in different resident vascular cell types. For instance, endothelial-specific knockout of GATA6 conveyed protection against ligation-induced neointimal hyperplasia through suppressing PDGF-B while SMC-specific GATA6 knockout promoted a synthetic SMC phenotype. Surprisingly, compared to GATA6, little is known about the function of GATA4, or a link between GATA4 and cyclin D1, in postnatal SMCs. The current article is the first demonstration of GATA4-mediated SMC proliferation in response to vascular injury. Further, GATA4 knockdown by shRNA completely abolished neointima formation in a femoral artery wire injury model, suggesting GATA4 may serve as a novel therapeutic target. Collectively these data suggest that the switch between GATA4 and GATA6 expression may be an important mechanism controlling the phenotypic state of SMCs in vascular development and in response to vascular injury. GATA4 is known to directly target promoters of cell cycle control genes in cardiomyocytes during heart development as embryonic GATA4 ablation or mutation blocked expression of cyclin D2 and cdk4 resulting in proliferation defects¹⁵. Here, Jeong, et. al.¹⁰ demonstrate that GATA4 directly activates cyclin D1 in SMCs and that cyclin D1 induction mediates the proliferative effects of GATA4, the first demonstration of such an axis in SMCs.

Figure 1. Model of nuclear FAK repression of SMC proliferation and neointimal hyperplasia.

(A). Vascular injury and growth factor/ECM-integrin signaling activate FAK kinase activity leading to GATA4 stability and GATA4-mediated SMC proliferation and neointimal hyperplasia. (B). Inhibition of FAK kinase activity with VH-4718 promotes FAK nuclear localization, GATA4 ubiquitination and degradation, and loss of GATA4-mediated cyclin D1-dependent SMC proliferation and neointimal hyperplasia.

Clinical Significance and Additional Questions.

FAK has emerged as a critical nodal signaling molecule that integrates ECM and growth factor signaling, which are critical to vascular disease progression. Subtle changes in expression or activity could have profound effects on normal vascular function that likely contribute to vascular disease progression. The findings presented here describe a novel endogenous inhibitory function for nuclear localized FAK that may have significant clinical impact leading to new and novel therapeutic approaches for the treatment of vascular disease. The data support that targeting FAK kinase activity is a novel approach to block SMC proliferation and neointima formation through nuclear FAK-mediated degradation of GATA4 and loss of GATA4-dependent cyclin D1 activity. Moving forward there are several unanswered questions. (a) What is the mechanism mediating nuclear FAK-dependent GATA4 proteasomal degradation? Does nuclear FAK directly interact with GATA4? And, if so, is this activity mediated through the FAK FERM domain similar to what was observed in endothelial cells? (b) The study was limited to SMC proliferation, yet additional functions contribute to pathological vascular remodeling (i.e. inflammation, ECM production). What effects, if any, does nuclear FAK exert on these functions? (c) What potential additional transcriptional regulators are also affected by nuclear FAK activity that might play a critical role in vascular disease progression? (d) What is the mechanism mediating FAK nuclear translocation and can this be targeted to prevent stimulus/disease-induced FAK nuclear-cytoplasmic shuttling as a mechanism to treat vascular disease? (e) Finally, nuclear FAK induced by FAK kinase inhibition in endothelial cells was shown by this group to negatively regulate FAK-mediated TNFα-induced inflammatory gene expression. Combined with the current study, these data highly support systemic or stent-based inhibition of FAK activity as a novel mechanism targeting multiple and cell-specific functions that promote vascular disease progression.