Aim: To elucidate the role of cytokine receptor-like factor 1 (CRLF1) in hepatic stellate cells and liver fibrosis.
Methods: Rat hepatic stellate cells (HSCs) were isolated by Nykodenz gradient centrifugation and activated by culturing in vitro. Differentially expressed genes in quiescent and culture activated HSCs were identified using microarrays. Injections of carbon tetrachloride (CCl(4)) for 4 wk were employed to induce liver fibrosis. The degree of fibrosis was assessed by Sirius red staining. Adenovirus expressing CRLF1 was injected through tail vein into mice to achieve overexpression of CRLF1 in the liver. The same adenovirus was used to overexpress CRLF1 in quiescent HSCs cultured in vitro. Expression of CRLF1, CLCF1 and ciliary neurotrophic factor receptor (CNTFR) in hepatic stellate cells and fibrotic livers was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and Western blotting. Expression of profibrotic cytokines and collagens was analyzed by the same method.
Results: CRLF1 is a secreted cytokine with unknown function. Human mutations suggested a role in development of autonomous nervous system and a role of CRLF1 in immune response was implied by its similarity to interleukin (IL)-6. Here we show that expression of CRLF1 was undetectable in quiescent HSCs and was highly upregulated in activated HSCs. Likewise, expression of CRLF1 was very low in normal livers, but was highly upregulated in fibrotic livers, where its expression correlated with the degree of fibrosis. A cofactor of CLRF1, cardiotrophin-like cytokine factor 1 (CLCF1), and the receptor which binds CRLF1/CLCF1 dimer, the CNTFR, were expressed to similar levels in quiescent and activated HSCs and in normal and fibrotic livers, indicating a constitutive expression. Overexpression of CLRF1 alone in the normal liver did not stimulate expression of profibrotic cytokines, suggesting that the factor itself is not pro-inflammatory. Ectopic expression in quiescent HSCs, however, retarded their activation into myofibroblasts and specifically decreased expression of type III collagen. Inhibition of type III collagen expression by CRLF1 was also seen in the whole liver. Our results suggest that CLRF1 is the only component of the CRLF1/CLCF1/CNTFR signaling system that is inducible by a profibrotic stimulus and that activation of this system by CLRF1 may regulate expression of type III collagen in fibrosis.
Conclusion: By regulating activation of HSCs and expression of type III collagen, CRLF1 may have an ability to change the composition of extracellular matrix in fibrosis.
Keywords: Cardiotrophin-like cytokine factor 1; Ciliary neurotrophic factor receptor; Cytokine receptor-like factor 1; Hepatic stellate cells; Liver fibrosis; Type III collagen.