Reduced Hepatic Stellate Cell Expression of Kruppel-Like Factor 6 Tumor Suppressor Isoforms Amplifies Fibrosis During Acute and Chronic Rodent Liver Injury

Abstract

Kruppel like factor 6 (KLF6), a zinc finger transcription factor and tumor suppressor, is induced as an immediate-early gene during hepatic stellate cell (HSC) activation. The paradoxical induction of a tumor suppressor in HSCs during proliferation led us to explore the biology of wild type KLF6 (KLF6WT) and its antagonistic, alternatively spliced isoform KLF6SV1 in cultured HSCs and animal models. Methods The animal models generated include a global heterozygous KLF6 mouse (Klf6 +/−), and transgenic mice expressing either hKLF6WT or hKLF6SV1 under the control of the Collagen α2 (I) promoter to drive HSC-specific gene expression following injury. Results The rat Klf6 transcript has multiple splice forms that are homologous to those of the human KLF6 gene. Following a transient increase, all rat Klf6 isoforms decreased in response to acute CCl4 liver injury, and culture-induced activation. After acute CCl4, Klf6 +/− mice developed significantly increased fibrosis and enhanced fibrogenic mRNA and protein expression. In contrast, HSC-specific transgenic mice over-expressing KLF6WT or KLF6SV1 developed significantly diminished fibrosis with reduced expression of fibrogenic genes. Chromatin IP, and qRT-PCR in mouse HSCs over-expressing KLF6WT demonstrated KLF6WT binding to GC boxes in promoters of Colα1 (I), Colα2 (I), and β-Pdgfr with reduced gene expression, consistent with transcriptional repression by KLF6. Stellate cells over-expressing either KLF6WT or KLF6SV1 were more susceptible to apoptotic stress based on PARP cleavage. Conclusion KLF6 reduces fibrogenic activity of HSCs via two distinct mechanisms, direct transcriptional repression of target fibrogenic genes and increased apoptosis of activated HSCs. These results suggest that following its initial induction, sustained downregulation of KLF6 in liver injury may allow de-repression of fibrogenic genes and decreased stellate cell clearance by inhibiting apoptosis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases necroinflammation and hepatic stellate cell activation but does not exacerbate experimental liver fibrosis in mice

“…On the other hand, TCDD has been shown to increase PAI-1 directly through binding of the AhR to a non-consensus XRE in the promoter region of the PAI-1 gene (Wilson et al , 2013). Interestingly, binding of the AhR to this non-consensus XRE involves the interaction of AhR with KLF6, a transcription factor that is known to repress fibrogenic gene expression in quiescent HSCs (Ghiassi-Nejad et al , 2013). It is conceivable that, when activated by TCDD, the AhR usurps KLF6 and prevents it from suppressing fibrogenic gene expression.…”

Hepatic stellate cells as key target in liver fibrosis

“…KLF6, a zinc finger transcription factor and tumor suppressor, directly represses collagen I and PDGFRβ expression and increases apoptosis of HSCs, and KLF6 haploinsufficiency increases fibrosis in rats [245]. Statins up-regulates KLF2 expression in sinusoidal endothelial cells, and induces vasoprotective gene expression, and quiescence of HSCs through a KLF2-nitric oxide-guanylate cyclase-mediated paracrine mechanism [246].…”

The characteristics of activated portal fibroblasts/myofibroblasts in liver fibrosis

“…Spp1, TIMP1, and others, and upregulate some of genes associated with quiescent phenotype in qHSCs, therefore reverting to a quiescent-like state (Kisseleva and Brenner, 2008; Troeger et al, 2012). Clearly, aHSC-derived myofibroblasts are attractive primary targets for anti-fibrotic therapy (Friedman and Bansal, 2006; Ghiassi-Nejad et al, 2013). Like aHSCs, aPFs /myofibroblasts can give rise to activated myofibroblasts that drive hepatic fibrosis and therefore may offer another anti-fibrotic therapy target particularly in the case of cholestatic injury.…”