Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor β–induced apoptosis†

Abstract

Hepatocytes in culture are a valuable tool to investigate mechanisms involved in the response of the liver to cytokines. However, it is well established that hepatocytes cultured on monolayers of dried stiff collagen dedifferentiate, losing specialized liver functions. In this study, we show that hepatocyte dedifferentiation is a reversible consequence of a specific signaling network constellation triggered by the extracellular matrix. A dried stiff collagen activates focal adhesion kinase (FAK) via Src, leading to activation of the Akt and extracellular signal-regulated kinase (ERK) 1/2 pathways. Akt causes resistance to transforming growth factor ␤ (TGF-␤)-induced apoptosis by antagonizing p38, whereas ERK1/2 signaling opens the route to epithelial-mesenchymal transition (EMT). Apoptosis resistance is reversible by inhibiting Akt or Src, and EMT can be abrogated by blocking the ERK1/2 pathway. In contrast to stiff collagen, a softer collagen gel does not activate FAK, keeping the hepatocytes in a state where they remain sensitive to TGF-␤-induced apoptosis and do not undergo EMT. In this culture system, inhibition of p38 as well as overexpression of constitutively active Akt causes apoptosis resistance, whereas constitutively active Ras induces EMT.

Toxicogenomics directory of chemically exposed human hepatocytes

“…However, if a gene induced by chemicals in vitro is also induced by the microenvironment of a diseased liver, this at least demonstrates that the involved mechanism is not a pure in vitro artifact. In vivo validation is of high relevance, since parts of the signaling network of cultivated hepatocytes are altered compared to hepatocytes in an intact liver, for example enhanced Akt activity mediating antiapoptotic mechanisms or increased MAK kinase signaling that causes features of epithelial-tomesenchymal transdifferentiation (Godoy et al 2009;; therefore, many responses observed in cultivated cells represent in vitro artifacts and should not be used for evaluation of chemicals. Only approximately 20 % of the chemically influenced genes in hepatocytes in vitro overlap with the genes altered in disease.…”

“…Moreover, there are insufficient studies demonstrating which of the responses of in vitro systems are relevant to the in vivo situation. For example, primary cultivated hepatocytes have been shown to become apoptosis resistant in culture, thereby possibly suppressing certain in vivo relevant responses (Godoy et al 2009(Godoy et al , 2010a. Moreover, cultivated hepatocytes are known to upregulate clusters of genes as a response to the isolation procedure and cultivation stress ).…”

Highlight report: acetaminophen hepatotoxicity

“…One of the most urgent clinical research fields seems to be the identification of biomarkers that predict more reliably whether a patient is in need of treatment Most likely the reason for resistance of cultivated hepatocytes to APAP is decreased expression CYP2E1 and further cytochrome P450 enzymes involved in APAP metabolic activation. It is well known that the isolation and cultivation stress lead to a rapid decrease in metabolizing enzymes in human hepatocytes (Godoy et al , 2015 and even more in rodents (Zellmer et al 2010;Heise et al 2012;Godoy et al 2009). Because of the relatively large discrepancy between APAP concentrations in vivo and higher concentrations chosen in many in vitro studies, it should be interpreted with caution, whether mechanisms observed in vitro at 10 mM and higher actually represent the in vivo situation.…”

Highlight report: cytoprotective signaling in toxicology

“…A further difficulty is that stress responses in cultivated cells may differ from those seen in vivo (Heise et al 2012). For example, cell culture conditions may induce more robust anti-apoptotic mechanisms than those generated in vivo (Godoy et al 2009. Therefore, the interpretation of stress responses with respect to adversity in vivo still remains a major challenge.…”