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The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen ␣1(I) mRNA, while activated HSCs contain about 60-to 70-fold more of this mRNA. The transcription rate of the collagen ␣1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen ␣1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen ␣1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen ␣1(I) mRNA 3 untranslated region (UTR). This sequence is localized 24 nucleotides 3 to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen ␣1(I) minigene and in stable transfections decreased the half-life of collagen ␣1(I) minigene mRNA. Binding to the collagen ␣1(I) 3 UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit ␣CP, which is also found in the complex involved in stabilization of ␣-globin mRNA. The auxiliary factors necessary to promote binding of ␣CP to the collagen 3 UTR are distinct from the factors necessary for binding to the ␣-globin sequence. Since ␣CP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen ␣1(I) mRNA 3 UTR.

Type I collagen is the most abundant protein in human body, produced by folding of two α1(I) and one α2(I) polypeptides into the triple helix. A conserved stem-loop structure is found in the 5' UTR of collagen mRNAs, encompassing the translation start codon. We cloned La ribonucleoprotein domain family, member 6 (LARP6) as the protein which binds the collagen 5' stem-loop in the sequence specific manner. LARP6 has a distinctive bipartite RNA binding domain, not found in other members of the La superfamily. LARP6 interacts with the two single stranded regions of 5' stemloop. The Kd for binding of LARP6 to the 5' stem-loop is 1.4 nM. LARP6 binds the 5' stem-loop in both, the nucleus and cytoplasm. In the cytoplasm, LARP6 does not associate with polysomes, however, overexpression of LARP6 blocks ribosomal loading on collagen mRNAs. Knocking down LARP6 by siRNA also decreased polysomal loading of collagen mRNAs, suggesting that it regulates translation. Collagen protein is synthesized at discrete regions of the endoplasmic reticulum (ER). We could reproduce this focal pattern of synthesis using collagen/GFP reporter protein, but only when the reporter was encoded by the mRNA with the 5' stem-loop and in the presence of LARP6. When the reporter was encoded by mRNA without the 5' stem-loop, or in absence of LARP6, it accumulated diffusely throughout the ER. This indicates that LARP6 activity is needed for focal synthesis of collagen polypeptides. We postulate that LARP6 dependent mechanism increases local concentration of collagen polypeptides for more efficient folding of the collagen heterotrimer.

Our findings show that transient human matrix metalloproteinase-1 overexpression in the liver effectively attenuates established fibrosis and induces hepatocyte proliferation.

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The stem-loop in the 5 untranslated region (UTR) of collagen ␣1(I) and ␣2(I) mRNAs (5SL) is the key element regulating their stability and translation. Stabilization of collagen mRNAs is the predominant mechanism for high collagen expression in fibrosis. LARP6 binds the 5SL of ␣1(I) and ␣2(I) mRNAs with high affinity. Here, we report that vimentin filaments associate with collagen mRNAs in a 5SL-and LARP6-dependent manner and stabilize collagen mRNAs. LARP6 interacts with vimentin filaments through its La domain and colocalizes with the filaments in vivo. Knockdown of LARP6 by small interfering RNA (siRNA) or mutation of the 5SL abrogates the interaction of collagen mRNAs with vimentin filaments. Vimentin knockout fibroblasts produce reduced amounts of type I collagen due to decreased stability of collagen ␣1(I) and ␣2(I) mRNAs. Disruption of vimentin filaments using a drug or by expression of dominant-negative desmin reduces type I collagen expression, primarily due to decreased stability of collagen mRNAs. RNA fluorescence in situ hybridization (FISH) experiments show that collagen ␣1(I) and ␣2(I) mRNAs are associated with vimentin filaments in vivo. Thus, vimentin filaments may play a role in the development of tissue fibrosis by stabilizing collagen mRNAs. This finding will serve as a rationale for targeting vimentin in the development of novel antifibrotic therapies.Fibroproliferative disorders are leading causes of morbidity and mortality globally (1,5,11,20,28,38) and pose an enormous threat to human health. There are no effective therapies for fibrotic diseases. Excessive production of type I collagen by activated fibroblasts and myofibroblasts is the hallmark of fibroproliferative disorders. Type I collagen is a heterotrimer composed of two ␣1(I) chains and one ␣2(I) chain (29). The increased collagen synthesis can be due to the increased rate of transcription of collagen genes, increased half-life of collagen mRNAs, and their enhanced translation (33,35,47,51). Increased collagen production by activated fibroblasts is primarily due to an increase in stability of collagen mRNAs (16). During activation of hepatic stellate cells, which synthesize collagen in liver fibrosis, a 16-fold prolongation of the half-life of collagen ␣1(I) mRNAs is primarily responsible for its 50-fold-increased expression (45). The transformation of fibroblasts into myofibroblasts is also associated with increased stability of collagen ␣1(I) mRNA (37). Transforming growth factor beta (TGF-␤), the most potent profibrotic cytokine, induces collagen synthesis by prolonging the half-life of collagen ␣1(I) mRNA (51). Thus, it is now well established that stability of collagen mRNAs is the predominant mechanism regulating collagen expression (30,40,41).Two cis-acting elements were implicated in regulating the stability of collagen mRNAs. In the 3Ј untranslated region (3Ј UTR) of collagen ␣1(I) mRNA, there is a cytosine-rich sequence that interacts with the ␣CP protein; this interaction stabilizes the mRNA (32, 34). In the 5Ј UTR of col...

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After liver injury, hepatic stellate cells (HSCs) undergo a process of activation with expression of smooth muscle ␣-actin (␣-SMA), an increased proliferation rate, and a dramatic increase in synthesis of type I collagen. The intracellular signaling mechanisms of activation and perpetuation of the activated phenotype in HSCs are largely unknown. In this study the role of the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, were evaluated in primary cultures of rat HSCs. The effect of JNK was assessed by using an adenovirus expressing a dominant negative form of transforming growth factor ␤ (TGF-␤)-activated kinase 1 (TAK1) (Ad5dnTAK1) and a new selective pharmacologic inhibitor SP600125. The effect of p38 was assessed with the selective pharmacologic inhibitor SB203580. These kinases were inhibited starting either in quiescent HSCs (culture day 1) or in activated HSCs (culture day 5). Although blocking TAK1/JNK and p38 decreased the expression of ␣-SMA protein in early stages of HSC activation, no effect was observed when TAK1/JNK or p38 were inhibited in activated HSCs. JNK inhibition increased and p38 inhibition decreased collagen ␣1(I) mRNA level as measured by RNase protection assays, with maximal effects observed in early stages of HSC activation. Furthermore, TAK1/JNK inhibition decreased HSC proliferation, whereas p38 inhibition led to an increased proliferation rate of HSCs, independently of its activation status. These results show novel roles for the TAK1/JNK pathway and p38 during HSC activation in culture. Despite similar activators of TAK1/JNK and p38, their functions in HSCs are distinct and opposed. (HEPATOLOGY 2001;34:953-963.)