Makoto Matsuyama

Sfrp1, Sfrp2, and Sfrp5 regulate the Wnt/β-catenin and the planar cell polarity pathways during early trunk formation in mouse

Sfrp is a secreted Wnt antagonist that directly interacts with Wnt ligand. We show here that inactivation of Sfrp1, Sfrp2, and Sfrp5 leads to fused somites formation in early‐somite mouse embryos, simultaneously resulting in defective convergent extension (CE), which causes severe shortening of the anteroposterior axis. These observations indicate the redundant roles of Sfrp1, Sfrp2, and Sfrp5 in early trunk formation. The roles of the Sfrps were genetically distinguished in terms of the regulation of Wnt pathways. Genetic analysis combining Sfrps mutants and Loop‐tail mice revealed the involvement of Sfrps in CE through the regulation of the planar cell polarity pathway. Furthermore, Dkk1‐deficient embryos carrying Sfrp1 homozygous and Sfrp2 heterozygous mutations display irregular somites and indistinct intersomitic boundaries, which indicates that Sfrps‐mediated inhibition of the Wnt/β‐catenin pathway is necessary for somitogenesis. Our results suggest that Sfrps regulation of the canonical and noncanonical pathways is essential for proper trunk formation. genesis 46:92–103, 2008.

Sfrp Controls Apicobasal Polarity and Oriented Cell Division in Developing Gut Epithelium

Epithelial tubular morphogenesis leading to alteration of organ shape has important physiological consequences. However, little is known regarding the mechanisms that govern epithelial tube morphogenesis. Here, we show that inactivation of Sfrp1 and Sfrp2 leads to reduction in fore-stomach length in mouse embryos, which is enhanced in the presence of the Sfrp5 mutation. In the mono-cell layer of fore-stomach epithelium, cell division is normally oriented along the cephalocaudal axis; in contrast, orientation diverges in the Sfrps-deficient fore-stomach. Cell growth and apoptosis are not affected in the Sfrps-deficient fore-stomach epithelium. Similarly, cell division orientation in fore-stomach epithelium diverges as a result of inactivation of either Stbm/Vangl2, an Fz/PCP component, or Wnt5a. These observations indicate that the oriented cell division, which is controlled by the Fz/PCP pathway, is one of essential components in fore-stomach morphogenesis. Additionally, the small intestine epithelium of Sfrps compound mutants fails to maintain proper apicobasal polarity; the defect was also observed in Wnt5a-inactivated small intestine. In relation to these findings, Sfrp1 physically interacts with Wnt5a and inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling controls oriented cell division and apicobasal polarity in the epithelium of developing gut.

Trichoplein and Aurora A block aberrant primary cilia assembly in proliferating cells

The trichoplein–AurA pathway must suppress primary cilia assembly in order for cells to exit G1.

Trichoplein controls microtubule anchoring at the centrosome by binding to Odf2 and ninein

The keratin cytoskeleton performs several functions in epithelial cells and provides regulated interaction sites for scaffold proteins, including trichoplein. Previously, we found that trichoplein was localized on keratin intermediate filaments and desmosomes in well-differentiated, non-dividing epithelia. Here, we report that trichoplein is widely expressed and has a major function in the correct localization of the centrosomal protein ninein in epithelial and non-epithelial cells. Immunocytochemical analysis also revealed that this protein is concentrated at the subdistal to medial zone of both mother and daughter centrioles. Trichoplein binds the centrosomal proteins Odf2 and ninein, which are localized at the distal to subdistal ends of the mother centriole. Trichoplein depletion abolished the recruitment of ninein, but not Odf2, specifically at the subdistal end. However, Odf2 depletion inhibited the recruitment of trichoplein to a mother centriole, whereas ninein depletion did not. In addition, the depletion of each molecule impaired MT anchoring at the centrosome. These results suggest that trichoplein has a crucial role in MT-anchoring activity at the centrosome in proliferating cells, probably through its complex formation with Odf2 and ninein.

A novel isoform of vinexin, vinexin gamma, regulates Sox9 gene expression through activation of MAPK cascade in mouse fetal gonad

Recent loss‐of‐function and gain‐of‐function studies have revealed that transcription factor Sox9 is required for testis formation by governing Sertoli cell differentiation, and thereafter regulating transcription of Sertoli marker genes. In the present study, we identified a novel isoform of Vinexin, which is expressed in somatic cells but not germ cells of sexually indifferent stages of fetal gonads. After the sex is determined, the expression continues in testicular Sertoli cells. Immunohistochemical analyses with a specific antibody to Vinexin indicated that Vinexin γ is localized in the cytoplasm. Functional studies with C3H10T1/2 cells showed that Vinexin γ acted as a scaffold protein to activate MEK and ERK through interaction with c‐Raf and ERK. Ultimately, Sox9 transcription was induced by Vinexin γ. This up‐regulation of Sox9 expression disappeared when the cells were treated with a specific MEK inhibitor, U0126. To determine the role of Vinexin γ during gonad formation, the gene was disrupted by targeted mutagenesis. The phenotype displayed by the mice indicated that ERK activation was decreased in the Vinexin γ−/– XY gonads, and Sox9 expression was down‐regulated. Thus, Vinexin γ seems to be implicated in regulation of Sox9 gene expression by modulating MAPK cascade in mouse fetal gonads.

P90 RSK arranges Chk1 in the nucleus for monitoring of genomic integrity during cell proliferation

P90 RSK, but not Akt/PKB, facilitates nuclear retention of Chk1 through Chk1–Ser-280 phosphorylation in response to serum stimulation. Chk1–Ser-280 phosphorylation is also elevated in a p90 RSK–dependent manner after UV irradiation and accelerates the Chk1 activation process (Ser-345 and Ser-296 phosphorylation on Chk1) after UV irradiation.

Defect of Mitotic Vimentin Phosphorylation Causes Microophthalmia and Cataract via Aneuploidy and Senescence in Lens Epithelial Cells

Background: Vimentin, an intermediate filament (IF) protein, is phosphorylated in mitosis. Results: Disruption of vimentin phosphorylation during cell division leads to chromosomal instability (CIN) and premature aging in mouse lens tissue. Conclusion: Our data document the first physiological importance of vimentin phosphorylation during mitosis for organogenesis and tissue homeostasis. Significance: Our data suggest a possible causal relationship between CIN and premature aging. Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIMSA/SA) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIMWT/SA) were indistinguishable from WT (VIMWT/WT) mice. In VIMSA/SA mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIMSA/SA, reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIMSA/SA, the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging.

Cytokinetic Failure-induced Tetraploidy Develops into Aneuploidy, Triggering Skin Aging in Phosphovimentin-deficient Mice.

Background: The fate of tetraploid cells in vivo remains largely unknown. Results: Tetraploid skin fibroblasts enter a new cell cycle and develop into aneuploid fibroblasts in phosphovimentin-deficient mice. Conclusion: These mice exhibited aging phenotypes in the skin. Significance: Our data suggest a possible causal relationship between tetraploidy and premature aging. Tetraploidy, a state in which cells have doubled chromosomal sets, is observed in ∼20% of solid tumors and is considered to frequently precede aneuploidy in carcinogenesis. Tetraploidy is also detected during terminal differentiation and represents a hallmark of aging. Most tetraploid cultured cells are arrested by p53 stabilization. However, the fate of tetraploid cells in vivo remains largely unknown. Here, we analyze the ability to repair wounds in the skin of phosphovimentin-deficient (VIMSA/SA) mice. Early into wound healing, subcutaneous fibroblasts failed to undergo cytokinesis, resulting in binucleate tetraploidy. Accordingly, the mRNA level of p21 (a p53-responsive gene) was elevated in a VIMSA/SA-specific manner. Disappearance of tetraploidy coincided with an increase in aneuploidy. Thereafter, senescence-related markers were significantly elevated in VIMSA/SA mice. Because our tetraploidy-prone mouse model also exhibited subcutaneous fat loss at the age of 14 months, another premature aging phenotype, our data suggest that following cytokinetic failure, a subset of tetraploid cells enters a new cell cycle and develops into aneuploid cells in vivo, which promote premature aging.

Nuclear Chk1 prevents premature mitotic entry

Chk1 inhibits the premature activation of the cyclin-B1–Cdk1. However, it remains controversial whether Chk1 inhibits Cdk1 in the centrosome or in the nucleus before the G2–M transition. In this study, we examined the specificity of the mouse monoclonal anti-Chk1 antibody DCS-310, with which the centrosome was stained. Conditional Chk1 knockout in mouse embryonic fibroblasts reduced nuclear but not centrosomal staining with DCS-310. In Chk1+/myc human colon adenocarcinoma (DLD-1) cells, Chk1 was detected in the nucleus but not in the centrosome using an anti-Myc antibody. Through the combination of protein array and RNAi technologies, we identified Ccdc-151 as a protein that crossreacted with DCS-310 on the centrosome. Mitotic entry was delayed by expression of the Chk1 mutant that localized in the nucleus, although forced immobilization of Chk1 to the centrosome had little impact on the timing of mitotic entry. These results suggest that nuclear but not centrosomal Chk1 contributes to correct timing of mitotic entry.

Secreted Frizzled-related protein 1 (Sfrp1) regulates the progression of renal fibrosis in a mouse model of obstructive nephropathy.

Background: Secreted Frizzled-related protein 1 is a secreted Wnt antagonist. Results: The kidneys from Sfrp1 knock-out mice showed significant increase in the renal fibrosis after unilateral ureteral obstruction. Conclusion: Deletion of Sfrp1 makes mice more susceptible to renal damage through non-canonical Wnt/PCP pathway. Significance: The relationship between kidney damage and Wnt/non-canonical pathway definitely opens a new field to study mechanisms of renal diseases. Renal fibrosis is responsible for progressive renal diseases that cause chronic renal failure. Sfrp1 (secreted Frizzled-related protein 1) is highly expressed in kidney, although little is known about connection between the protein and renal diseases. Here, we focused on Sfrp1 to investigate its roles in renal fibrosis using a mouse model of unilateral ureteral obstruction (UUO). In wild-type mice, the expression of Sfrp1 protein was markedly increased after UUO. The kidneys from Sfrp1 knock-out mice showed significant increase in expression of myofibrobast markers, α-smooth muscle actin (αSMA). Sfrp1 deficiency also increased protein levels of the fibroblast genes, vimentin, and decreased those of the epithelial genes, E-cadherin, indicated that enhanced epithelial-to-mesenchymal transition. There was no difference in the levels of canonical Wnt signaling; rather, the levels of phosphorylated c-Jun and JNK were more increased in the Sfrp1−/− obstructed kidney. Moreover, the apoptotic cell population was significantly elevated in the obstructed kidneys from Sfrp1−/− mice following UUO but was slightly increased in those from wild-type mice. These results indicate that Sfrp1 is required for inhibition of renal damage through the non-canonical Wnt/PCP pathway.