MethodsUUO. Smad3-null (Smad3 ex8/ex8 ) mice were generated as described (13) and were used at 6-8 weeks of age and 20-30 g in body weight. In mice sedated by general anes-

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

Abstract-The klotho gene, originally identified by insertional mutagenesis in mice, suppresses the expression of multiple aging-associated phenotypes. This gene is predominantly expressed in the kidney. Recent studies have shown that expression of renal klotho gene is regulated in animal models of metabolic diseases and in humans with chronic renal failure. However, little is known about the mechanisms and the physiological relevance of the regulation of the expression of the klotho gene in the kidney in some diseased conditions. In the present study, we first investigated the role of angiotensin II in the regulation of renal klotho gene expression. Long-term infusion of angiotensin II downregulated renal klotho gene expression at both the mRNA and protein levels. This angiotensin II-induced renal klotho downregulation was an angiotensin type 1 receptor-dependent but pressor-independent event. Adenovirus harboring mouse klotho gene (ad-klotho, 3.3ϫ10 10 plaque forming units) was also intravenously administered immediately before starting angiotensin II infusion in some rats. This resulted in a robust induction of Klotho protein in the liver at day 4, which was still detectable 14 days after the gene transfer. T he klotho gene, identified by insertional mutagenesis in mice, is a suppressor of the expression of multiple aging phenotypes similar to age-related diseases in humans, such as arteriosclerosis, osteoporosis, infertility, pulmonary emphysema, and short lifespan. 1 Interestingly, expression of klotho mRNA in the kidney can be only faintly detected in the prenatal rat, and it is markedly augmented after 4 days of age. 2 Although the klotho gene has a role in phenotypic alterations in various organs, expression of klotho mRNA is predominantly observed in the kidney, 1 suggesting that the Klotho protein or its metabolites may function as humoral factors. Recent studies have shown that expression of renal klotho gene is regulated in animals 2 and in humans 3 in some diseased conditions. At present, however, the mechanism regulating klotho gene expression is poorly understood.In the present study, we have investigated the role of angiotensin (Ang) II in the regulation of renal klotho gene expression. In addition, to clarify the possible physiological role of the klotho gene in the Ang II-infused rats, exogenous klotho gene was delivered into Ang II-infused rats, and functional and histological changes in the kidney were analyzed. Methods Animal ModelsThe experiments were performed in accordance with the guidelines and practices established by the Animal Center for Biomedical Research, University of Tokyo, Faculty of Medicine. The rat Ang II hypertension model was induced in male Sprague-Dawley rats (Nippon Bio-Supply Center, Tokyo, Japan) by the continuous infusion of [Val 5 ]-Ang II (Sigma) at a dose of 0.7 mg/kg per day via an osmotic minipump (Alza) as described previously. 4 In some experiments, the selective angiotensin type 1 receptor antagonist losartan (25 mg/kg per day; a gift from Merck, Rahway, NJ) ...

MethodsUUO. Smad3-null (Smad3 ex8/ex8 ) mice were generated as described (13) and were used at 6-8 weeks of age and 20-30 g in body weight. In mice sedated by general anes-

SummaryKeratin filaments form cytoskeletal networks in epithelial cells. Dynamic rearrangement of keratin filament networks is required for epithelial cells to perform cellular processes such as cell migration and polarization; however, the mechanism governing keratin filament rearrangement remains unclear. Here, we describe a novel mechanism of keratin cytoskeleton organization mediated by casein kinase Ia (CK-1a) and a newly identified keratin-associated protein, FAM83H. Knockdown of FAM83H induces keratin filament bundling, whereas overexpression of FAM83H disassembles keratin filaments, suggesting that FAM83H regulates the filamentous state of keratins. Intriguingly, keratin filament bundling is concomitant with the dissociation of CK-1a from keratin filaments, whereas aberrant speckle-like localization of CK-1a is observed concomitantly with keratin filament disassembly. Furthermore, CK-1a inhibition, similar to FAM83H knockdown, causes keratin filament bundling and reverses keratin filament disassembly induced by FAM83H overexpression, suggesting that CK-1a mediates FAM83H-dependent reorganization of keratin filaments. Because the N-terminal region of FAM83H interacts with CK-1a and the C-terminal region interacts with keratins, FAM83H might tether CK-1a to keratins. Colorectal cancer tissue also shows keratin filament disassembly accompanied with FAM83H overexpression and aberrant CK-1a localization, and FAM83H-overexpressing cancer cells exhibit loss or alteration of epithelial cell polarity. Importantly, knockdown of FAM83H inhibits cell migration accompanied by keratin cytoskeleton rearrangement in colorectal cancer cells. These results suggest that keratin cytoskeleton organization is regulated by FAM83H-mediated recruitment of CK-1a to keratins, and that keratin filament disassembly caused by overexpression of FAM83H and aberrant localization of CK-1a could contribute to the progression of colorectal cancer.