The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia, including the epidermis. Here we generated claudin-1–deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1–deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (and also claudin-4–positive) TJs. Interestingly, in the wild-type epidermis these occludin-positive TJs efficiently prevented the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface, whereas in the claudin-1–deficient epidermis the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis and that these TJs are crucial for the barrier function of the mammalian skin.
Smad7 negatively regulates transforming growth factor (TGF)-b superfamily signaling by binding to activated type I receptors, thereby preventing the phosphorylation of receptor-regulated Smads (R-Smads), as well as by recruiting HECT-type E3 ubiquitin ligases to degrade type I receptors through a ubiquitin-dependent mechanism. To elucidate the regulatory mechanisms of TGF-b signaling, we searched for novel members of proteins that interact with Smad7 using a yeast two-hybrid system. One of the proteins identified was the WW domain-containing protein 1 (WWP1) that is structurally related to Smad ubiquitin regulatory factors (Smurfs), E3 ubiquitin ligases for Smads and TGF-b superfamily receptors. Using a TGF-b-responsive reporter in mammalian cells, we found that WWP1 inhibited transcriptional activities induced by TGF-b. Similar to Smurfs, WWP1 associated with Smad7 and induced its nuclear export, and enhanced binding of Smad7 to TGF-b type I receptor to cause ubiquitination and degradation of the receptor. Consistent with these results, WWP1 inhibited phosphorylation of Smad2 induced by TGF-b. WWP1 thus negatively regulates TGF-b signaling in cooperation with Smad7. However, unlike Smurfs, WWP1 failed to ubiquitinate R-Smads and SnoN. Importantly, WWP1 and Smurfs were expressed in distinct patterns in human tissues and carcinoma cell lines, suggesting unique pathophysiological roles of WWP1 and Smurfs.
Although attention has focused on the chemopreventive action of retinoic acid (RA) in hepatocarcinogenesis, the functional role of RA in the liver has yet to be clarified. To explore the role of RA in the liver, we developed transgenic mice expressing RA receptor (RAR) ␣-dominant negative form in hepatocytes using albumin promoter and enhancer. At 4 months of age, the RAR ␣-dominant negative form transgenic mice developed microvesicular steatosis and spotty focal necrosis. Mitochondrial -oxidation activity of fatty acids and expression of its related enzymes, including VLCAD, LCAD, and HCD, were down-regulated; on the other hand, peroxisomal -oxidation and its related enzymes, including AOX and BFE, were up-regulated. Expression of cytochrome p4504a10, cytochrome p4504a12, and cytochrome p4504a14 was increased, suggesting that -oxidation of fatty acids in microsomes was accelerated. In addition, formation of H 2 O 2 and 8-hydroxy-2-deoxyguanosine was increased. After 12 months of age, these mice developed hepatocellular carcinoma and adenoma of the liver. The incidence of tumor formation increased with age. Expression of -catenin and cyclin D1 was enhanced and the TCF-4/-catenin complex was increased, whereas the RAR ␣/ -catenin complex was decreased. Feeding on a high-RA diet reversed histological and biochemical abnormalities and inhibited the occurrence of liver tumors. These results suggest that hepatic loss of RA function leads to the development of steatohepatitis and liver tumors. In conclusion, RA plays an important role in preventing hepatocarcinogenesis in association with fatty acid metabolism and Wnt signaling. (HEPATOLOGY 2004;40:366-375.)
Purpose: To optimize the implantable collamer lens (ICL) sizing method using anterior segment optical coherence tomography (AS-OCT). Setting: Nagoya Eye Clinic, Nagoya, Japan. Design: Interventional case series. Methods: A stepwise multiple regression analysis was performed using the optimal ICL size as a dependent variable and preoperative AS-OCT parameters as explanatory variables for 81 eyes of 41 patients, and the NK-formula version 2 (NK-formula V2) was obtained. Thereafter, 68 eyes of 42 patients were implanted with the ICLs selected as closest to the optimal ICL size calculated by the NK-formula V2. At 3 months postoperatively, the achieved vault was measured by AS-OCT to evaluate the optimization of the sizing method. Results: The anterior chamber width (ACW) and crystalline lens rise were selected as significant parameters for the regression model (R 2 = 0.61, P < .001), as with the previous NK-formula. Of the 68 eyes, 36 patients/62 eyes (91.2%), 5 patients/5 eyes (7.3%), and 1 patient/1 eye (1.5%) were in the moderate, high, and low vault categories, respectively. In the 68 eyes, the vault showed no correlation with the optimal ICL size (R = 0.0185, P = .269), whereas the vault showed a negative correlation with the optimal ICL size in both the 12.6 mm ICL subgroup (R = −0.409, P = .0088) and the 13.2 mm ICL subgroup (R = −0.818, P = .0013). Conclusions: This optimization approach showed excellent ability to select an appropriate ICL to be implanted regardless of the value of other ocular parameters and age, except ACW.
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