Physiological ranges of matrix rigidity modulate primary mouse hepatocyte function in part through hepatocyte nuclear factor 4 alpha

Abstract

Matrix rigidity has important effects on cell behavior and is increased during liver fibrosis; however, its effect on primary hepatocyte function is unknown. We hypothesized that increased matrix rigidity in fibrotic livers would activate mechanotransduction in hepatocytes and lead to inhibition of hepatic-specific functions. To determine the physiologically relevant ranges of matrix stiffness at the cellular level, we performed detailed atomic force microscopy analysis across liver lobules from normal and fibrotic livers. We determined that normal liver matrix stiffness was around 150Pa and increased to 1–6kPa in areas near fibrillar collagen deposition in fibrotic livers. In vitro culture of primary hepatocytes on collagen matrix of tunable rigidity demonstrated that fibrotic levels of matrix stiffness had profound effects on cytoskeletal tension and significantly inhibited hepatocyte-specific functions. Normal liver stiffness maintained functional gene regulation by hepatocyte nuclear factor 4 alpha (HNF4α) whereas fibrotic matrix stiffness inhibited the HNF4α transcriptional network. Fibrotic levels of matrix stiffness activated mechanotransduction in primary hepatocytes through focal adhesion kinase (FAK). In addition, blockade of the Rho/Rho-associated protein kinase (ROCK) pathway rescued HNF4α expression from hepatocytes cultured on stiff matrix. Conclusion Fibrotic levels of matrix stiffness significantly inhibit hepatocyte-specific functions in part by inhibiting the HNF4α transcriptional network mediated through the Rho/ROCK pathway. Increased appreciation of the role of matrix rigidity in modulating hepatocyte function will advance our understanding of the mechanisms of hepatocyte dysfunction in liver cirrhosis and spur development of novel treatments for chronic liver disease.

HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity

“…Loss of adhesion is one of the first events in plasma-induced toxicity, and anchorage proteins, such as E-cadherin and the beta1-integrin receptor are known to protect against hepatocyte dedifferentiation and apoptosis [28][29][30][31]. In addition, matrix rigidity is known to limit hepatocyte differentiation, in part through transcription factor HNF4A [32].…”

Force-dependent breaching of the basement membrane

“…Liver fibrosis is associated with increased liver tissue stiffness, as determined by non-invasive imaging modalities such as ultrasound-based transient and shear-wave elastography [40] and magnetic resonance elastography [41]. Atomic force microscopy measurements demonstrate that normal liver matrix is around 150Pa and is increased to 1-6kPa in experimental models of liver fibrosis [42]. Increased matrix stiffness activates stellate cells and portal fibroblasts, the primary cell types responsible for abnormal ECM deposition during the development of fibrosis [43,44].…”

“…Importantly, increased matrix rigidity also has a direct inhibitory effect on hepatocyte function by activating mechano-signal transduction through FAK and decreasing expression of hepatocyte nuclear factor 4 alpha (HNF4α), a master transcriptional regulator of hepatic function. Moreover, expression of HNF4α in primary hepatocytes on stiff matrix can be rescued by inhibition of ROCK signaling, indicating a critical role of the FAK-ROCK signaling network in modulating hepatocyte function in response to tissue mechanics [42]. In addition to regulating function, HNF4α is also a tumor suppressor.…”

“…In addition to regulating function, HNF4α is also a tumor suppressor. HNF4α expression is reduced in fibrotic livers and forced re-expression of HNF4α can attenuate the progression of fibrosis [42,45,46]. Reduced expression of HNF4α expression promotes hepatocyte proliferation [47] and activation of mesenchymal genes [48].…”

Reply

“…We thank Gortzen and Trebicka for their interest in our work (1). Studies suggest that mechanical force regulates RhoA mainly at the level of activity, by increasing the amount of activated GTP-bound RhoA, modulating guanine nucleotide exchange factors, and/or coupling GTP-RhoA to Rho-kinase (ROCK) (2–4).…”