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Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis. Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation. Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology.

During amphibian intestinal remodeling, thyroid hormone (TH) induces adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian one. We have previously shown that hyaluronan (HA) is newly synthesized and plays an essential role in the development of the stem cells via its major receptor CD44 in the Xenopus laevis intestine. We here focused on HA synthase (HAS) and examined how the expression of HAS family genes is regulated during natural and TH-induced metamorphosis. Our quantitative RT-PCR analysis indicated that the mRNA expression of HAS2 and HAS3, but not that of HAS1 and HAS-rs, a unique Xenopus HAS-related sequence, is upregulated concomitantly with the development of adult epithelial primordia consisting of the stem/progenitor cells during the metamorphic climax. In addition, our in situ hybridization analysis indicated that the HAS3 mRNA is specifically expressed in the adult epithelial primordia, whereas HAS2 mRNA is expressed in both the adult epithelial primordia and nearby connective tissue cells during this period. Furthermore, by treating X. laevis tadpoles with 4-methylumbelliferone, a HA synthesis inhibitor, we have experimentally shown that inhibition of HA synthesis leads to suppression of TH-upregulated expression of leucine-rich repeat-containing G protein-coupled 5 (LGR5), an intestinal stem cell marker, CD44, HAS2, HAS3, and gelatinase A in vivo. These findings suggest that HA newly synthesized by HAS2 and/or HAS3 is required for intestinal stem cell development through a positive feedback loop and is involved in the formation of the stem cell niche during metamorphosis.

Anti‐proinflammatory cytokine therapies against interleukin (IL)‐6, tumor necrosis factor (TNF)‐α, and IL‐1 are major advancements in treating inflammatory diseases, especially rheumatoid arthritis. Such therapies are mainly performed by injection of antibodies against cytokines or cytokine receptors. We initially found that the glycolytic inhibitor 2‐deoxy‐d‐glucose (2‐DG), a simple monosaccharide, attenuated cellular responses to IL‐6 by inhibiting N‐linked glycosylation of the IL‐6 receptor gp130. Aglycoforms of gp130 did not bind to IL‐6 or activate downstream intracellular signals that included Janus kinases. 2‐DG completely inhibited dextran sodium sulfate‐induced colitis, a mouse model for inflammatory bowel disease, and alleviated laminarin‐induced arthritis in the SKG mouse, an experimental model for human rheumatoid arthritis. These diseases have been shown to be partially dependent on IL‐6. We also found that 2‐DG inhibited signals for other proinflammatory cytokines such as TNF‐α, IL‐1β, and interferon ‐γ, and accordingly, prevented death by another inflammatory disease, lipopolysaccharide (LPS) shock. Furthermore, 2‐DG prevented LPS shock, a model for a cytokine storm, and LPS‐induced pulmonary inflammation, a model for acute respiratory distress syndrome of coronavirus disease 2019 (COVID‐19). These results suggest that targeted therapies that inhibit cytokine receptor glycosylation are effective for treatment of various inflammatory diseases. The glycolytic inhibitor 2‐deoxy‐D‐glucose attenuated cellular responses to proinflammatory cytokines by inhibiting N‐linked glycosylation of their receptors and alleviated the signs and symptoms of mouse models for inflammatory bowel disease, rheumatoid arthritis, cytokine storm, and acute respiratory distress syndrome.

The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established in the so-called postembryonic developmental period in mammals when endogenous thyroid hormone (T3) levels are high. The T3-dependent metamorphosis in anurans like Xenopus laevis resembles the mammalian postembryonic development and offers a unique opportunity to study how the adult stem cells are developed. The tadpole intestine is predominantly a monolayer of larval epithelial cells. During metamorphosis, the larval epithelial cells undergo apoptosis and, concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a well-established stem cell marker in the adult mouse intestinal crypt. Here we have cloned and analyzed the spatiotemporal expression profile of LGR5 gene during frog metamorphosis. We show that the two duplicated LGR5 genes in Xenopus laevis and the LGR5 gene in Xenopus tropicalis are highly homologous to the LGR5 in other vertebrates. The expression of LGR5 is induced in the limb, tail, and intestine by T3 during metamorphosis. More importantly, LGR5 mRNA is localized to the developing adult epithelial stem cells of the intestine. These results suggest that LGR5-expressing cells are the stem/progenitor cells of the adult intestine and that LGR5 plays a role in the development and/or maintenance of the adult intestinal stem cells during postembryonic development in vertebrates.

During amphibian intestinal remodeling, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian epithelium. To clarify molecular mechanisms underlying adult epithelial development, we here focus on TH response genes that are associated with the canonical Wnt pathway. Our quantitative reverse transcription plus polymerase chain reaction and immunohistochemical analyses indicate that all of the genes examined, including β-catenin, c-Myc and secreted frizzle-related protein 2 (SFRP2), are up-regulated in Xenopus laevis intestine during both natural and TH-induced metamorphosis. Moreover, immunoreactivity for nuclear β-catenin becomes detectable in adult stem cells from the start of their appearance and then increases in intensity in adult epithelial primordia derived from the stem cells, which actively proliferate and coexpress Wnt target genes c-Myc and LGR5. These expression profiles strongly suggest the involvement of the canonical Wnt pathway in the maintenance and/or proliferation of adult stem/progenitor cells. More importantly, by using organ cultures of the tadpole intestine, we have experimentally shown that the addition of exogenous SFRP2 protein to the culture medium promotes cell proliferation of the adult epithelial primordia, whereas inhibition of endogenous SFRP2 by its antibody suppresses their proliferation. The inhibition of SFRP2 suppresses larval epithelial changes in shape from simple columnar to stem-cell-like roundish cells, resulting in the failure of epithelial dedifferentiation. Thus, TH-up-regulated SFRP2 in the postembryonic intestine promotes adult stem cell development, possibly by acting as an agonist of both canonical and non-canonical Wnt signaling.

Background/Aims:The mechanisms that regulate the size-related morphologies of various blood vessels from the aorta to capillary vessels are still poorly understood. In this study, we evaluate the involvement of regulator of calcineurin 1 (RCAN1), a regulatory protein in the calcineurin/NFAT signal transduction pathway, in vascular morphology to gain further insight into these mechanisms. Methods and Results: We first generated 2 types of vasculature in vitro from the same source of human umbilical vein endothelial cells by fibrin gel assay. We found that RCAN1 was significantly upregulated in large vessels with low branching frequencies when compared with small vessels with high branching frequencies. Next, to clarify whether RCAN1 regulates the branching of blood vessels in vivo, we injected RCAN1 mRNA into fertilized Xenopus laevis eggs. Overexpression of RCAN1 decreased the number of branching points that sprouted from intersomitic vessels during X. laevis angiogenesis. In addition, coexpression of calcineurin A, a target of RCAN1, could rescue RCAN1-suppressed vascular branching. Conclusions: These results provide in vivo evidence of RCAN1-regulated vascular branching which may play a role in the patterning of morphologically different vasculature.

During amphibian intestinal remodeling, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian epithelium. To clarify molecular mechanisms underlying adult epithelial development, we here focus on TH response genes that are associated with the canonical Wnt pathway. Our quantitative reverse transcription plus polymerase chain reaction and immunohistochemical analyses indicate that all of the genes examined, including [beta]-catenin, c-Myc and secreted frizzle-related protein 2 (SFRP2), are up-regulated in Xenopus laevis intestine during both natural and TH-induced metamorphosis. Moreover, immunoreactivity for nuclear [beta]-catenin becomes detectable in adult stem cells from the start of their appearance and then increases in intensity in adult epithelial primordia derived from the stem cells, which actively proliferate and coexpress Wnt target genes c-Myc and LGR5. These expression profiles strongly suggest the involvement of the canonical Wnt pathway in the maintenance and/or proliferation of adult stem/progenitor cells. More importantly, by using organ cultures of the tadpole intestine, we have experimentally shown that the addition of exogenous SFRP2 protein to the culture medium promotes cell proliferation of the adult epithelial primordia, whereas inhibition of endogenous SFRP2 by its antibody suppresses their proliferation. The inhibition of SFRP2 suppresses larval epithelial changes in shape from simple columnar to stem-cell-like roundish cells, resulting in the failure of epithelial dedifferentiation. Thus, TH-up-regulated SFRP2 in the postembryonic intestine promotes adult stem cell development, possibly by acting as an agonist of both canonical and non-canonical Wnt signaling.

Twin and family studies had shown that genetic factors are important determinants of bone mass. Multiple genes might be involved. One candidate gene, the reversion-induced LIM gene ( RIL ), is a PDZ and LIM-domain-containing protein and has been localized within the cytokine cluster of chromosome 5 (5q31.1). In a genetic study of 370 adult Japanese women, we investigated the correlation between radial bone mineral density (BMD) and a genetic variation (−3333T→C) of the 5'-flanking region of RIL gene. A significant association was identified between the RIL variation −3333T→C and radial BMD ( r =0.15, P =0.003). The variation of the RIL locus may be an important determinant of osteoporosis.

Among multiple factors influencing osteoporosis, genetic variations involved in bone-mineral metabolism can affect risks predisposing to the disease onset. Here, we studied single-nucleotide polymorphisms (SNPs) in the pro-opiomelanocortin ( POMC ) gene for possible association with bone mineral density (BMD) among 384 adult Japanese women and observed significant correlation between adjusted BMD and three SNPs in the promoter region ( r >0.14, p <0.01). The most significant correlation was observed for −2353G/A ( r =−0.16, p =0.002); homozygous carriers of the major (G) allele had the highest BMD (0.405±0.054 g/cm 2 ) while heterozygous carriers were intermediate (0.390±0.053 g/cm 2 ) and homozygous A-allele carriers had the lowest BMDs (0.369±0.048 g/cm 2 ). Although no association was detected between these SNPs and body weight or body mass index (BMI), significant association was detected between the −2313A/C genotype and plasma total cholesterol level ( r =−0.12, p =0.019). We propose that POMC is among the likely susceptibility genes for osteoporosis and may also be involved in dyslipidemia.

.  Osteoporosis is believed to result from interplay among multiple environmental and genetic determinants, including factors that regulate bone-mineral density (BMD). Recent quantitative trait locus analysis in human suggested a possible involvement of chromosomal region 1p36.2–p36.3 for determination of BMD. The brain natriuretic peptide ( BNP , also named NPPB ) gene lies within this candidate region for BMD determination. Overexpression of the BNP resulted in skeletal overgrowth in transgenic mice. Association analysis between nucleotide variations of the BNP gene and radial BMD in 378 Japanese postmenopausal women revealed a significant association of the –381T/C variation of the BNP gene with radial BMD ( r = 0.17, P = 0.01). Homozygous T-allele carriers had the lowest BMD values (0.395 ± 0.056 g/cm 2 ), homozygous C-allele carriers had the highest (0.429 ± 0.051 g/cm 2 ), and heterozygous individuals had intermediate radial BMD values (0.405 ± 0.048 g/cm 2 ), indicating a dosage effect. Accelerated bone loss also correlated with the –381 T allele in a 5-year follow-up study ( r = 0.21, P = 0.017). These results suggest that variation of BNP may be an important determinant of postmenopausal osteoporosis, in part through the mechanism of accelerated postmenopausal bone loss.