Mitsuko Kajita

Origin of the adult intestinal stem cells induced by thyroid hormone in Xenopus laevis

In the amphibian intestine during metamorphosis, de novo stem cells generate the adult epithelium analogous to the mammalian counterpart. Interestingly, to date the exact origin of these stem cells remains to be determined, making intestinal metamorphosis a unique model to study development of adult organ‐specific stem cells. Here, to determine their origin, we made use of transgenic Xenopus tadpoles expressing green fluorescent protein (GFP) for recombinant organ cultures. The larval epithelium separated from the wild‐type (Wt) or GFP transgenic (Tg) intestine before metamorphic climax was recombined with homologous and heterologous nonepithelial tissues and was cultivated in the presence of thyroid hormone, the causative agent of metamorphosis. In all kinds of recombinant intestine, adult progenitor cells expressing markers for intestinal stem cells such as sonic hedgehog became detectable and then differentiated into the adult epithelium expressing intestinal fatty acid binding‐protein, a marker for absorptive cells. Notably, whenever the epithelium was derived from Tg intestine, both the adult progenitor/stem cells and their differentiated cells expressed GFP, whereas neither of them expressed GFP in the Wt‐derived epithelium. Our results provide direct evidence that stem cells that generate the adult intestinal epithelium originate from the larval epithelium, through thyroid hormone‐induced dedifferentiation.— Ishizuya‐Oka, A.,Hasebe, T., Buchholz, D. R., Kajita, M., Fu, L., Shi, Y.‐B. The origin of the adult intestinal stem cells induced by thyroid hormone in Xenopus laevis. FASEB J. 23, 2568–2575 (2009)

Association of Molecular Variants, Haplotypes, and Linkage Disequilibrium Within the Human Vitamin D‐Binding Protein (DBP) Gene With Postmenopausal Bone Mineral Density

Possible contribution of vitamin D‐binding protein (DBP) gene for determination of BMD was tested by characterizing 13 SNPs in 384 adult Japanese women. When the effect of a specific single SNP was tested, five SNPs (−39C>T, IVS1+827C>T, IVS1+1916C>T, IVS1‐1154A>G, and IVS11+1097G>C) correlated with BMD significantly at various levels. The chromosomal dosage of one haplotype (T‐C‐C‐G‐T‐C in −39C>T, IVS1+827C>T, IVS1+1916C>T, IVS1‐1154A>G, D432E, and IVS11+1097G>C) displayed significant correlation with adjusted radial BMD (r = 0.15, p = 0.008; n = 331). Multiple regression analyses revealed a most significant correlation with the combination of IVS1+827C>T and D432E (r2 = 0.029, p = 0.005). These results indicate a complex combined effect of several SNPs within the DBP gene that might underlie susceptibility to low radial BMD and osteoporosis.

Association of Multiple Nucleotide Variations in the Pituitary Glutaminyl Cyclase Gene (QPCT) With Low Radial BMD in Adult Women

Correlation between 13 genetic variations of the glutaminyl‐peptide cyclotransferase gene and adjusted aBMD was tested among 384 adult women. Among 13 variations with strong linkage disequilibrium, R54W showed a prominent association (p = 0.0003), which was more striking when examined among 309 elder subjects (≥50 years; p = 0.0001). Contribution for postmenopausal bone loss was suggested.

Spatio-Temporal Expression Profile of Stem Cell- Associated Gene LGR5 in the Intestine during Thyroid Hormone-Dependent Metamorphosis in Xenopus laevis

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Overestimated frequency of a possible emphysema-susceptibility allele when microsomal epoxide hydrolase is genotyped by the conventional polymerase chain reaction-based method

AbstractA recent association study suggested that the His113 variant of microsomal epoxide hydrolase (mEPHX) may confer a risk for development of emphysema, presumably by increasing susceptibility to smoking injury. Before considering a possible role of this enzyme in pulmonary disease, we attempted to characterize the genetic polymorphism further. The Tyr/His113 polymorphism within exon 3 of mEPHX was initially examined in 62 healthy individuals by conventional methods involving polymerase chain reaction (PCR)-based determination of a restriction fragment length polymorphism (RFLP). Genomic nucleotide sequences, including the polymorphic site and the downstream primer sequence, were further analyzed in 95 unrelated, healthy Japanese volunteers by single-stranded conformation polymorphism (SSCP) analysis and direct sequencing. Genotyping by the first method (PCR-RFLP) revealed that the allelic distribution in our test population apparently deviated from Hardy-Weinberg equilibrium. Sequence analysis showed that a synonymous nucleotide substitution, AAG to AAA (Lys119), was located just within the published primer site. The AAA at codon 119 was present only in alleles with Tyr113, and its frequency reached 0.31 in our panel of 190 Japanese alleles. This substitution potentially hampered PCR amplification because of the nucleotide mismatch, with the result that the frequency of the Tyr113 variation was underestimated. The frequency of His113, a possible emphysema susceptibility allele of the mEPHX gene, was thus overestimated when human DNA samples were genotyped in the conventional way. Depending on the population(s) tested, this anomaly could represent a pitfall for PCR-based association studies.

Expression profiles of the duplicated matrix metalloproteinase-9 genes suggest their different roles in apoptosis of larval intestinal epithelial cells during Xenopus laevis metamorphosis.

Matrix metalloproteinases (MMPs) play a pivotal role in development and/or pathogenesis through degrading extracellular matrix (ECM) components. We have previously shown that Xenopus MMP‐9 gene is duplicated. To assess possible roles of MMP‐9 and MMP‐9TH in X. laevis intestinal remodeling, we here analyzed their expression profiles by in situ hybridization and show that their expression is transiently up‐regulated during thyroid hormone‐dependent metamorphosis. Of interest, MMP‐9TH mRNA is strictly localized in the connective tissue and most highly expressed just beneath the larval epithelium that begins to undergo apoptosis. On the other hand, cells expressing MMP‐9 mRNA become first detectable in the connective tissue and then, after the start of epithelial apoptosis, also in the larval epithelium. These results strongly suggest that MMP‐9TH is responsible in the larval epithelial apoptosis through degrading ECM components in the basal lamina, whereas MMP‐9 is involved in the removal of dying epithelial cells during amphibian intestinal remodeling. Developmental Dynamics 236:2338–2345, 2007.

Association of a Trp16Ser variation in the gonadotropin releasing hormone signal peptide with bone mineral density, revealed by SNP-dependent PCR typing

Osteoporosis is believed to result from interplay among multiple environmental and genetic determinants, including factors that regulate bone mineral density (BMD). Among those factors, adequate estrogen is essential for achievement of peak bone mass as well as for postmenopausal maintenance of skeletal homeostasis. Gonadotropin-releasing hormone (GnRH) from the hypothalamus is the primary determinant in the hypothalamic-pituitary-gonadal feedback system. In genetic studies of 384 postmenopausal Japanese women, we found a significant association between BMD and an amino acid variation (Trp16Ser) located within the signal peptide of GnRH (r = 0.143, P = 0.005). These results were achieved by genotyping all subjects using a newly developed SNP-dependent PCR method. This automated, high-throughput, and inexpensive procedure is suitable for typing large numbers of samples. BMD was lowest among 16Ser/Ser homozygotes, highest among 16Trp/Trp homozygotes, and intermediate among heterozygotes. A case-control study involving 125 osteoporosis patients and 92 healthy controls revealed a significant association between the presence of a 16Ser GnRH allele and affected status (chi(2) = 4.74, P = 0.041). The results suggested that variation of the GnRH signal peptide may be an important risk factor for postmenopausal osteoporosis.

Thirteen single-nucleotide polymorphisms in the human osteopontin gene identified by sequencing of the entire gene in Japanese individuals

AbstractOsteopontin (OPN) is one of the major noncollagenous bone matrix proteins produced by osteoblasts and osteoclasts. We systematically surveyed the entire structure of the OPN gene for single-nucleotide polymorphisms (SNPs) by directly sequencing 48 alleles derived from 24 unrelated Japanese individuals. We identified 13 SNPs in the OPN gene. Ten polymorphisms were identified in introns 1, 3, and 5; 2 in the coding region of exons 6 and 7; and 1 in the 3′ untranslated region of exon 7. Allele frequencies for some of the polymorphisms were significantly different from those reported in the United States National Center for Biotechnology Information (NCBI) dbSNP database. These polymorphisms will be useful in genetic studies to evaluate the role of OPN proteins in bone metabolism.

Thyroid hormone-up-regulated hedgehog interacting protein is involved in larval-to-adult intestinal remodeling by regulating sonic hedgehog signaling pathway in Xenopus laevis.

Sonic hedgehog (Shh) was previously shown to be involved in the larval‐to‐adult remodeling of the Xenopus laevis intestine. While Shh is transcriptionally regulated by thyroid hormone (TH), the posttranscriptional regulation of Shh signaling during intestinal remodeling is largely unknown. In the present study, we focused on a role of the pan‐hedgehog inhibitor, hedgehog interacting protein (Hip), in the spatiotemporal regulation of Shh signaling. Using real‐time reverse transcriptase‐polymerase chain reaction and in situ hybridization, we show that Hip expression is transiently up‐regulated during both natural and TH‐induced metamorphosis and that Hip mRNA is localized in the connective tissue adjacent to the adult epithelial primordia expressing Shh. Interestingly, the expression of bone morphogenetic protein‐4, a Shh target gene, is hardly detectable where Hip is strongly expressed. Finally, we demonstrate that Hip binds to the N‐terminal fragment of processed Shh in vivo, suggesting that Hip suppresses Shh signaling through sequestering Shh. Developmental Dynamics 237:3006–3015, 2008.

Co-segregation of elevated LDL with a novel mutation (D92K) of the LDL receptor in a kindred with multiple lipoprotein abnormalities

AbstractFactors predisposing to the phenotypic features of familial combined hyperlipidemia have not been clearly defined. In the course of investigating familial coronary artery disease in Utah, we identified a three-generation family in which multiple members were affected with type IIa hyperlipoproteinemia (HLP IIa), type IIb hyperlipoproteinemia (HLP IIb), or type IV hyperlipoproteinemia (HLP IV). Because several family members had relatively severe low-density lipoprotein (LDL) cholesterol elevation, in order to dissect the possible contribution to the plasma lipoprotein abnormalities in this pedigree, we identified a novel point mutation in the low-density lipoprotein receptor (LDLR) gene, a G-to-A transition at nucleotide position 337 in exon 4. This change substituted lysine for glutamic acid at codon 92 (D92K) of the LDL receptor. By means of mutant allele-specific amplification we determined that the mutation co-segregated with elevated cholesterol and LDL cholesterol in the plasma of family members with HLP IIa and HLP IIb, but not with the elevated plasma triglycerides seen in HLP IIb and HLP IV patients. Thus, in families with apparent familial combined hyperlipidemia, a defective LDLR allele and other genetic or environmental factors that elevate plasma triglycerides may account for the multiple lipid phenotypes observed in this kindred.