Kazuo Ikeda

MicroRNA-221/222 upregulation indicates the activation of stellate cells and the progression of liver fibrosis

Background MicroRNAs (miRNAs) are important in hepatic pathophysiology and the development of liver cancer. Objective To explore miRNAs that are regulated with the progression of liver fibrosis caused by chronic liver disease. Design The regulated miRNAs in human livers infected with hepatitis C virus were identified by microarray analysis. Their expression in human livers with non-alcoholic steatohepatitis, mouse livers from two fibrosis models and cultured stellate cells was validated by real-time RT-PCR. The regulation of miR-222 expression in stellate cells by nuclear factor kappa B (NF-κB) was assayed. Finally, the effects of an miR-222 precursor or inhibitor on the expression of cyclin-dependent kinase inhibitor 1B (CDKN1B) and the growth of LX-2 cells were determined. Results It was found that miR-199a-5p/199a-3p and miR-221/222 were upregulated in the human liver in a fibrosis progression–dependent manner. Among these miRNAs, miR-221/222 were upregulated in LX-2 cells and increased during the course of culture-dependent activation of mouse primary stellate cells, in a manner similar to the expression of α1(I) collagen and α-smooth muscle actin mRNAs. The expression of miR-221/222 increased in mouse models of liver fibrosis. In contrast, an NF-κB inhibitor significantly suppressed the miR-222 induction that was stimulated in culture by transforming growth factor α or tumour necrosis factor α. Although overexpression or downregulation of miR-222 failed to regulate the growth of LX-2 cells, miR-222 bound to the CDKN1B 3′UTR and regulated the expression of the corresponding protein. Conclusion miR-221/222 may be new markers for stellate cell activation and liver fibrosis progression.

Negligible Contribution of Bone Marrow-Derived Cells to Collagen Production During Hepatic Fibrogenesis in Mice

BACKGROUND & AIMS Recent studies have reported that bone marrow (BM)-derived cells migrating into fibrotic liver tissue exhibit a myofibroblast-like phenotype and may participate in the progression of liver fibrosis. However, their contribution to collagen production has not been fully verified yet. We revisited this issue by using 2 mechanistically distinct liver fibrosis models introduced into transgenic collagen reporter mice and their BM recipients. METHODS BM of wild-type mice was replaced by cells obtained from transgenic animals harboring tissue-specific enhancer/promoter sequences of alpha2(I) collagen gene (COL1A2) linked to enhanced green fluorescent protein (EGFP) or firefly luciferase (LUC) gene. Liver fibrosis was introduced into those mice by repeated carbon tetrachloride injections or ligation of the common bile duct. Activation of COL1A2 promoter was assessed by confocal microscopic examination detecting EGFP signals and luciferase assays of liver homogenates. RESULTS The tissue-specific COL1A2 enhancer/promoter was activated in hepatic stellate cells following a single carbon tetrachloride injection or during primary culture on plastic. A large number of EGFP-positive collagen-expressing cells were observed in liver tissue of transgenic COL1A2/EGFP mice in both liver fibrosis models. In contrast, there were few EGFP-positive BM-derived collagen-producing cells detected in fibrotic liver tissue of COL1A2/EGFP recipients. Luciferase assays of liver tissues from COL1A2/LUC-recipient mice further indicated that BM-derived cells produced little collagen in response to fibrogenic stimuli. CONCLUSIONS By using a specific and sensitive experimental system, which detects exclusively BM-derived collagen-producing cells, we conclude an unexpectedly limited role of BM-derived cells in collagen production during hepatic fibrogenesis.

Down-regulation of cyclin E1 expression by microRNA-195 accounts for interferon-β-induced inhibition of hepatic stellate cell proliferation.

Recent studies have suggested that interferons (IFNs) have an antifibrotic effect in the liver independent of their antiviral effect although its detailed mechanism remains largely unknown. Some microRNAs have been reported to regulate pathophysiological activities of hepatic stellate cells (HSCs). We performed analyses of the antiproliferative effects of IFNs in HSCs with special regard to microRNA‐195 (miR‐195). We found that miR‐195 was prominently down‐regulated in the proliferative phase of primary‐cultured mouse HSCs. Supporting this fact, IFN‐β induced miR‐195 expression and inhibited the cell proliferation by delaying their G1 to S phase cell cycle progression in human HSC line LX‐2. IFN‐β down‐regulated cyclin E1 and up‐regulated p21 mRNA levels in LX‐2 cells. Luciferase reporter assay revealed the direct interaction of miR‐195 with the cyclin E1 3′UTR. Overexpression of miR‐195 lowered cyclin E1 mRNA and protein expression levels, increased p21 mRNA and protein expression levels, and inhibited cell proliferation in LX‐2 cells. Moreover miR‐195 inhibition restored cyclin E1 levels that were down‐regulated by IFN‐β. In conclusion, IFN‐β inhibited the proliferation of LX‐2 cells by delaying cell cycle progression in G1 to S phase, partially through the down‐regulation of cyclin E1 and up‐regulation of p21. IFN‐induced miR‐195 was involved in these processes. These observations reveal a new mechanistic aspect of the antifibrotic effect of IFNs in the liver. J. Cell. Physiol. 226: 2535–2542, 2011.

Promotion of Liver and Lung Tumorigenesis in DEN-Treated Cytoglobin-Deficient Mice

Cytoglobin (Cygb) is a recently discovered vertebrate globin with molecular characteristics that are similar to myoglobin. To study the biological function of Cygb in vivo, we generated Cygb knockout mice and investigated their susceptibility to N,N-diethylnitrosamine (DEN)-induced tumorigenesis. Four-week-old male mice were administered DEN in drinking water at a dose of 25 ppm for 25 weeks or 0.05 ppm for 36 weeks. Cygb deficiency promoted the DEN-induced development of liver and lung tumors. All Cygb(+/-) and Cygb(-/-) mice treated with 25-ppm DEN exhibited liver tumors, compared with 44.4% of their wild-type counterparts. Lung tumors were present only in Cygb-deficient mice. More than 40% of Cygb(-/-) mice developed liver and lung tumors at the nontoxic dose of DEN (0.05 ppm), which did not induce tumors in wild-type mice. Cygb loss was associated with increased cancer cell proliferation, elevated extracellular signal-regulated kinase and Akt activation, overexpression of IL-1β, IL-6, Tnfα, and Tgfβ3 mRNAs, and hepatic collagen accumulation. Cygb-deficient mice also exhibited increased nitrotyrosine formation and dysregulated expression of cancer-related genes (cyclin D2, p53, Pak1, Src, Cdkn2a, and Cebpa). These results suggest that Cygb deficiency induces susceptibility to cancer development in the liver and lungs of mice exposed to DEN. Thus, globins such as Cygb will shed new light on the biological features of organ carcinogenesis.

Corneal wound healing in an osteopontin-deficient mouse.

PURPOSE To investigate the effects of loss of osteopontin (OPN) in the healing of the injured cornea in mice. Cell culture study was also conducted to clarify the effects of OPN in fibroblast behaviors. METHODS Ocular fibroblasts from wild-type (WT) and OPN-null (KO) mice were used to study the role of OPN on cell behavior. The effect of the lack of OPN on corneal wound healing was evaluated in mice. RESULTS In cell culture, OPN is involved in cell adhesion and in the migration of ocular fibroblasts. Adhesion of the corneal epithelial cell line was not affected by exogenous OPN. OPN was upregulated in a healing, injured mouse cornea. Loss of OPN did not affect epithelial healing after simple epithelial debridement. Healing of an incision injury in cornea was delayed, with less appearance of myofibroblasts and transforming growth factor beta1 expression in a KO mouse than in a WT mouse. The absence of OPN promoted tissue destruction after an alkali burn, resulting in a higher incidence of corneal perforation in KO mice than in WT mice. CONCLUSIONS OPN modulates wound healing-related fibroblast behavior and is required to restore the physiological structure of the cornea after wound healing.

Pitavastatin inhibits hepatic steatosis and fibrosis in non‐alcoholic steatohepatitis model rats

Aim:  Non‐alcoholic steatohepatitis (NASH) may progress to liver cirrhosis, and NASH patients with liver cirrhosis are at risk of developing hepatocellular carcinoma. Statins, 3‐hydroxy‐3‐methyglutaryl‐coenzyme A reductase inhibitors, are well known to reduce low‐density lipoprotein cholesterol and reduce the incidence of coronary heart disease and other major vascular events by anti‐inflammatory and antifibrotic effects, and antiproliferative properties in colorectal cancers have also been reported. Recently, statins have been reported to improve hepatic steatosis; however, the effect on fibrosis is controversial.

Dynamic relationship of focal contacts and hemidesmosome protein complexes in live cells.

Epidermal cells adhere to the basement membrane zone through cell-matrix junctions termed hemidesmosomes. During wound healing, hemidesmosomes are disassembled to allow keratinocytes to move over wound sites. Such movement is mediated by both hemidesmosome protein complexes (HPCs) and focal contacts (FCs). In this study, we analyzed the interaction between HPCs and FCs in live HaCat cells expressing yellow fluorescent protein (YFP)-tagged beta4 integrin and cyan fluorescent protein (CFP)-tagged alpha-actinin as markers of HPCs and FCs, respectively. In HaCat cells migrating to repopulate wounds, FC proteins cluster rapidly in the direction of the wound. HPC assembly then follows and the newly formed HPCs occupy sites vacated by the disassembled FCs. HPC dynamics are dramatically reduced, and HaCat cells cease migration upon treatment with reagents that affect FC integrity/function. Upon treatment with reagents that destabilize HPCs, the dynamics of FCs in HaCat cells at the edges of wounds are enhanced, although FC assembly is irregular and the migration of the cells is aberrant. We also show that the complex interaction between hemidesmosomes and FCs in keratinocytes is myosin dependent and requires energy. In summary, we suggest that HPCs and FCs dynamics are tightly co-regulated in keratinocytes undergoing migration during wound healing.

Induction of tropomyosin during hepatic stellate cell activation and the progression of liver fibrosis

The activation of hepatic stellate cells (HSCs) is a cue to initiate liver fibrosis. Activated stellate cells acquire contractile activity similar to pericytes and myofibroblasts in other organs by inducing the contractile machinery of cytoskeletons such as smooth muscle α-actin (α-SMA), a well-known marker of activated stellate cells, and actin-binding proteins. We further show herein the expression of tropomyosin in rat HSCs in the course of their activation during primary culture and liver tissue damaged by thioacetamide intoxication. In immunoblot analysis, tropomyosin became detectable in an early stage of the primary culture of rat stellate cells in a manner similar to the expression of α-SMA and platelet-derived growth factor receptor-β. Tropomyosin was found to be colocalized with α-SMA on fluorescent immunocytochemistry. At the liver tissue level, an increased expression of tropomyosin was observed by immunoblot analysis and immunohistochemistry along the septum of fibrosis, where α-SMA was enriched. These results strongly suggest that tropomyosin is a new marker of activated stellate cells and may serve as a useful diagnostic marker of liver fibrosis.

Laminin-511, inducer of hair growth, is down-regulated and its suppressor in hair growth, laminin-332 up-regulated in chemotherapy-induced alopecia

BACKGROUND Chemotherapy-induced alopecia (CIA) has a devastating cosmetic effect, especially in the young. Recent data indicate that two major basement membrane components (laminin-332 and -511) of the skin have opposing effects on hair growth. OBJECTIVE In this study, we examined the role and localization of laminin-332 and -511 in CIA. METHODS We examined the expression of laminin-332 and -511 during the dystrophic catagen form of CIA induced in C57BL/6 mice by cyclophosphamide (CYP) treatment. RESULTS Our data indicate that both laminin-332 and its receptor alpha 6 beta 4 integrin are up-regulated (both quantitatively and spatially) after mid to late dystrophic catagen around the outer root sheath (ORS) in the lower third of hair follicles in CIA. This up-regulation also occurs at the transcriptional level. In contrast, laminin-511 is down-regulated after mid dystrophic catagen at the protein level, with transcriptional inactivation of laminin-511 occurring transiently at the early dystrophic catagen stage in both epidermal and ORS keratinocytes. Laminin-511 expression correlates with expression of alpha 3 integrin in CIA and we also demonstrate that laminin-511 can up-regulate the activity of the alpha 3 integrin promoter in cultured keratinocytes. Injection of a laminin-511 rich protein extract, but not recombinant laminin-332, in the back skin of mice delays hair loss in CYP-induced CIA. CONCLUSIONS We propose that abrupt hair loss in CIA is, at least in part, caused by down-regulation of laminin-511 and up-regulation of laminin-332 at the transcriptional and translational levels.