Regulation of MT1-MMP and MMP-2 by Leptin in Cardiac Fibroblasts Involves Rho/ROCK-Dependent Actin Cytoskeletal Reorganization and Leads to Enhanced Cell Migration

Abstract

Altered leptin action has been implicated in the pathophysiology of heart failure in obesity, a hallmark of which is extracellular matrix remodeling. Here, we characterize the direct influence of leptin on matrix metalloproteinase (MMP) activity in primary adult rat cardiac fibroblasts and focus on elucidating the molecular mechanisms responsible. Leptin increased expression and cell surface localization of membrane type 1 (MT1)-MMP, measured by cell surface biotinylation assay and antibody-based colorimetric detection of an exofacial epitope in intact cells. Coimmunoprecipitation analysis showed that leptin also induced the formation of a cluster of differentiation 44/MT1-MMP complex. Qualitative analysis using rhodamine-conjugated phalloidin immunofluorescence indicated that leptin stimulated actin cytoskeletal reorganization and enhanced stress fiber formation. Hence, we analyzed activation of Ras homolog gene family (Rho), member A GTPase activity and found a rapid increase in response to leptin that corresponded with increased phosphorylation of cofilin. Quantitative analysis of cytoskeleton reorganization upon separation of globular and filamentous actin by differential centrifugation confirmed the significant increase in filamentous to globular actin ratio in response to leptin, which was prevented by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated coiled-coil-forming protein kinase (ROCK) (Y-27632). Inhibition of Rho or ROCK also attenuated leptin-stimulated increases in cell surface MT1-MMP content. Pro-MMP-2 is a known MT1-MMP substrate, and we observed that enhanced cell surface MT1-MMP in response to leptin resulted in enhanced extracellular activation of pro-MMP-2 measured by gelatin zymography, which was again attenuated by inhibition of Rho or ROCK. Using wound scratch assays, we observed enhanced cell migration, but not proliferation, measured by 5-bromo2'-deoxy-uridine incorporation, in response to leptin, again via a Rho-dependent signaling mechanism. Our results suggest that leptin regulates myocardial matrix remodeling by regulating the cell surface localization of MT1-MMP in adult cardiac fibroblasts via Rho/ROCK-dependent actin cytoskeleton reorganization. Subsequent pro-MMP-2 activation then contributes to stimulation of cell migration.

Effects of linagliptin and liraglutide on glucose- and angiotensin II-induced collagen formation and cytoskeleton degradation in cardiac fibroblasts in vitro

“…The disassembly of F-actin cytoskeleton inhibits migration of cardiac fibroblasts [20] , and the inhibition of F-actin assembly has also been proved to inhibit the fibroblast-myofibroblast transition [21] . An in vivo study has also shown that actin cytoskeleton remodeling attenuates myocardial fibrosis following myocardial infarction in rats [22] .…”

EP3 receptor deficiency attenuates pulmonary hypertension through suppression of Rho/TGF-β1 signaling

“…Activation of RhoA/ROCK signaling is known to be involved in the regulation of actin cytoskeletal remodeling (40), which has recently been recognized to mediate the trafficking of membrane type 1-MMP (MT1-MMP) to the cell surface (41)(42)(43). MT1-MMP is a transmembrane protease that cleaves extracellular pro-MMP-2.…”

“…These observations indicate that EP3α/β-mediated actin cytoskeletal remodeling regulates intracellular MT1-MMP transportation through activation of Rho GTPase. CD44, a hyaluronic acid receptor, is believed to interact with the hemopexin-like domain of MT1-MMP (41) and to link MT1-MMP with actin filaments through direct CD44 binding to F-actin (41,43). As such, leptin mediates myocardial matrix remodeling by regulating the cell-surface location of MT1-MMP in cardiac fibroblasts via Rho GTPase-mediated actin polymerization, which facilitates the development of heart failure in obesity (43,64).…”

“…CD44, a hyaluronic acid receptor, is believed to interact with the hemopexin-like domain of MT1-MMP (41) and to link MT1-MMP with actin filaments through direct CD44 binding to F-actin (41,43). As such, leptin mediates myocardial matrix remodeling by regulating the cell-surface location of MT1-MMP in cardiac fibroblasts via Rho GTPase-mediated actin polymerization, which facilitates the development of heart failure in obesity (43,64). Thus, EP3α/β activation, which stimulates the MMP-2/TGF-β1-signaling cascade in the PASMC response to hypoxic stress, induces Rho/ROCK-dependent actin cytoskeletal reorganization, an event that may be implicated in PAH pathogenesis.…”

Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)

“…(i) MMP14 modulates glycolysis and is regulated by leptin, the famous obese gene product (38). (ii) Insulin receptor substrate 4 (IRS4), in addition to its known function in insulin-Akt-mTOR signaling, directly binds to and modulates signaling through the leptin receptor (39).…”