Interplay of Matrix Stiffness and c-SRC in Hepatic Fibrosis

Abstract

Introduction: In liver fibrosis activation of hepatic stellate cells (HSC) comprises phenotypical change into profibrotic and myofibroplastic cells with increased contraction and secretion of extracellular matrix (ECM) proteins. The small GTPase RhoA orchestrates cytoskeleton formation, migration, and mobility via non-receptor tyrosine-protein kinase c-SRC (cellular sarcoma) in different cells. Furthermore, RhoA and its downstream effector Rho-kinase also play a crucial role in hepatic stellate cells and hepatic fibrogenesis. Matrix stiffness promotes HSC activation via cytoskeleton modulation. This study investigated the interaction of c-SRC and RhoA under different matrix stiffness conditions.Methods: Liver fibrosis was induced in rats using bile duct ligation (BDL), thioacetamide (TAA) or carbon tetrachloride (CCl4) models. mRNA levels of albumin, PDGF-R, RHOA, COL1A1, and αSMA were analyzed via qRT-PCR. Western Blots using phospho-specific antibodies against p-c-SRC418 and p-c-SRC530 analyzed the levels of activating and inactivating c-SRC, respectively. LX2 cells and hepatocytes were cultured on acrylamide gels of 1 and 12 kPa or on plastic to mimic non-fibrotic, fibrotic, or cirrhotic environments then exposed to SRC-inhibitor PP2. Overexpression of RhoA was performed by transfection using RhoA-plasmids. Additionally, samples from cirrhotic patients and controls were collected at liver transplantations and tumor resections were analyzed for RhoA and c-SRC protein expression by Western Blot.Results: Transcription of albumin and RhoA was decreased, whereas transcription and activation of c-SRC was increased in hepatocytes cultured on 12 kPa compared to 1 kPa gels. LX2 cells cultured on 12 kPa gels showed upregulation of RHOA, COL1A1, and αSMA mRNA levels. Inhibition of c-SRC by PP2 in LX2 cells led to an increase in COL1A1 and αSMA most prominently in 12 kPa gels. In LX2 cells with RhoA overexpression, c-SRC inhibition by PP2 failed to improve fibrosis. RhoA expression was significantly elevated in human and experimental liver fibrosis, while c-SRC was inactivated.Conclusions: This study shows that c-SRC is inactive in activated myofibroblast-like HSC in liver cirrhosis. Inactivation of c-SRC is mediated by a crosstalk with RhoA upon hepatic stellate cell activation and fibrosis progression.

Src Inhibition Attenuates Liver Fibrosis by Preventing Hepatic Stellate Cell Activation and Decreasing Connective Tissue Growth Factor

“…However, few studies have investigated the role of Src in liver fibrosis. A recent report showed that phosphorylation of Src at Y416 and Y530 is decreased and increased during liver fibrosis, respectively [12]. However, the current study demonstrated that phosphorylation of Src at Y416 was increased during the activation of primary HSCs and in liver tissues of TAA-injected mice.…”

“…Src was originally identified as an oncogene and it is expressed in human tumors; however, it is also ubiquitously expressed in all cell types. Src phosphorylates the signaling proteins STAT3, AKT, and epidermal growth factor receptor (EGFR), thereby regulating various biological activities, including cell survival, proliferation, and migration [10][11][12]. The activation of Src causes pulmonary and renal fibrosis [13,14].…”

Hepatic stellate cells as key target in liver fibrosis

“…These changes serve as mechanical stimuli to activate HSCs at least partially via integrin signaling pathways, forming positive feedback loops [50, 51]. LX-2 cells cultured on acrylamide gels with 12 kPa pressure, mimicking increased tissue stiffness in fibrotic liver, show higher collagen I expression via RHOA upregulation, which is suppressed by c-Src [52]. Primary HSCs cultured in hydrogels stiffened or softening in situ over time revealed time-course dynamic transcriptional changes during the process of culture activation and regression, respectively [53, 54].…”

Sinusoidal communication in liver fibrosis and regeneration

“…When cultured on stiff rather than soft collagen lattices PMF increase their expression of fibrillin-1 [40], and HSC become highly activated loosing their lipid droplets and expressing high levels of α-SMA [41], demonstrating that the stiffness of the matrix has significant functional relevance for the contractile non-parenchymal cells. Interestingly, a recent paper further depicts the underlying mechanisms partly responsible for such activation revealing a negative regulation between RhoA and the cytosolic tyrosine kinase c-SCR [42]. …”