Stress that impairs endoplasmic reticulum (ER) function leads to an accumulation of unfolded or misfolded proteins in the ER (ER stress) and triggers the unfolded protein response (UPR). Recent studies suggest that ER stress is involved in idiopathic pulmonary fibrosis (IPF). The present study was undertaken to determine the role of ER stress on myofibroblastic differentiation of fibroblasts. Fibroblasts in fibroblastic foci of IPF showed immunoreactivity for GRP78. To determine the role of ER stress on α-smooth muscle actin (α-SMA) and collagen type I expression in fibroblasts, mouse and human lung fibroblasts were treated with TGF-β1, and expression of ER stress-related proteins, α-SMA, and collagen type I was analyzed by Western blotting. TGF-β1 significantly increased expression of GRP78, XBP-1, and ATF6α, which was accompanied by increases in α-SMA and collagen type I expression in mouse and human fibroblasts. A chemical chaperone, 4-PBA, suppressed TGF-β1-induced UPR and α-SMA and collagen type I induction. We also showed that TGF-β1-induced UPR was mediated through the reactive oxygen species generation. Our study provides the first evidence implicating the UPR in myofibroblastic differentiation during fibrosis. These findings of the role of ER stress and chemical chaperones in pulmonary fibrosis may improve our understanding of the pathogenesis of IPF.

Found in inflammatory zone (FIZZ) 2, also known as resistin-like molecule (RELM)-β, belongs to a novel cysteine-rich secreted protein family named FIZZ/RELM. Its function is unclear, but a closely related family member, FIZZ1, has profibrotic activities. The human ortholog of rodent FIZZ1 has not been identified, but human FIZZ2 has significant sequence homology to both rodent FIZZ2 (59%) and FIZZ1 (50%). Given the greater homology to rodent FIZZ2, analyzing the role of FIZZ2 in a rodent model of bleomycin-induced pulmonary fibrosis would be of greater potential relevance to human fibrotic lung disease. The results showed that FIZZ2 was highly induced in lungs of rodents with bleomycin-induced pulmonary fibrosis and of human patients with idiopathic pulmonary fibrosis. FIZZ2 expression was induced in rodent and human lung epithelial cells by Th2 cytokines, which was mediated via STAT6 signaling. The FIZZ2 induction in murine lungs was found to be essential for pulmonary fibrosis, as FIZZ2 deficiency significantly suppressed pulmonary fibrosis and associated enhanced extracellular matrix and cytokine gene expression. In vitro analysis indicated that FIZZ2 could stimulate type I collagen and α-smooth muscle actin expression in lung fibroblasts. Furthermore, FIZZ2 was shown to have chemoattractant activity for bone marrow (BM) cells, especially BM-derived CD11c+ dendritic cells. Notably, lung recruitment of BM-derived cells was impaired in FIZZ2 knockout mice. These findings suggest that FIZZ2 is a Th2-associated multifunctional mediator with potentially important roles in the pathogenesis of fibrotic lung diseases.

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A single administration of complete Freund's adjuvant (CFA), type 1 carrageenan (Car), or silica 7, 2, and 2 days, respectively, before infection with a low dose (1 x 10(2) plaque-forming units/mouse) of encephalomyocarditis D (EMC-D) virus resulted in a significant increase in the incidence of diabetes in SJL/J mice (100%) compared with untreated EMC-D virus-infected mice (40%). Peritoneal macrophages were isolated from uninfected SJL/J mice, which had been treated once with silica, and transferred into SJL/J mice 2 days before low-dose EMC-D infection. Approximately 90% of the mice became diabetic, whereas 30% of mice that received virus alone became diabetic. The depletion of macrophages by treatment with the combined anti-Mac-1 and anti-Mac-2 monoclonal antibodies after a single administration of CFA, Car, or silica resulted in almost complete prevention of beta-cell destruction in EMC-D virus-infected mice. Furthermore, none of the mice in which macrophages were depleted by long-term treatment with silica and 10% of the mice treated with Car before virus infection became diabetic. On the basis of these observations, we conclude that macrophages are directly involved in the destruction of beta-cells, leading to the development of clinical diabetes in EMC-D virus-infected mice.

PurposeThe success of basic molecular research using biospecimens strongly depends on the quality of the specimen. In this study, we evaluated the effects of delayed freezing time on the stability of DNA and RNA in fresh frozen tissue from patients with colorectal cancer.MethodsTissues were frozen at 10, 30, 60, and 90 minutes after extirpation of colorectal cancer in 20 cases. Absorbance ratio of 260 to 280 nm (A260/A280) and agarose gel electrophoresis were evaluated. In addition, the RNA integrity number (RIN) was assayed for the analysis of the RNA integrity.ResultsRegardless of delayed freezing time, all DNA and RNA samples revealed A260/A280 ratios of more than 1.9, and all DNA samples showed a discrete, high-molecular-weight band on agarose gel electrophoresis. The RINs were 7.53 ± 2.04, 6.70 ± 1.88, 6.47 ± 2.58, and 4.22 ± 2.34 at 10, 30, 60, and 90 minutes, respectively. Though the concentration of RNA was not affected by delayed freezing, the RNA integrity was decreased with increasing delayed freezing time.ConclusionAccording to the RIN results, we recommend that the collection of colorectal cancer tissue should be done within 10 minutes for studies requiring RNA of high quality and within 30 minutes for usual RNA studies.