Geun-Shik Lee

Phenotype of a calbindin-D9k gene knockout is compensated for by the induction of other calcium transporter genes in a mouse model.

CaBP‐9k may be involved in the active calcium absorption and embryo implantation. Although we generated CaBP‐9k KO mice to explore its function, no distinct phenotypes were observed in these KO mice. It can be hypothesized that TRPV5 and 6 and plasma membrane calcium ATPase 1b may play a role in the regulation of calcium transport to compensate CaBP‐9k deficiency in its KO model.

Expression of human Calbindin-D9k correlated with age, vitamin D receptor and blood calcium level in the gastrointestinal tissues

OBJECTIVEnCalbindin-D(9k) (CaBP-9k) has been thought to be one of the important factors of calcium absorption and metabolism in the intestine. The understanding of CaBP-9k role in the gastrointestinal tissues is important in human in relation with aging and gender, and whether there is any relationship with mineral homeostasis and risk for the development of age-related calcium disorders such as osteoporosis.nnnDESIGN AND METHODSnThe expression of CaBP-9k and vitamin D receptor (VDR) was analyzed in the human gastrointestinal tissues with different ages and gender. In addition, the correlation among CaBP-9k expression, age and calcium concentration in blood was elucidated in these tissues.nnnRESULTSnA significant increase in the expression of CaBP-9k mRNA was observed in the bulb and 2(nd) portion of duodenum over age in both men and women. However, no significant age-associated change in VDR mRNA level was detected in the duodenum. The concentration of blood calcium level decreased with age in both men and women.nnnCONCLUSIONnThe duodenal CaBP-9k may be not involved in calcium absorption in these tissues. The functional significance of the increase in the intestinal CaBP-9k expression with age is not currently clear and requires further investigation.

Mouse calbindin-D9k gene expression in the uterus during late pregnancy and lactation

Abstract Calbindin-D 9k (CaBP-9k) is a cytosolic calcium binding protein mainly expressed in duodenum, placenta and uterus. In order to understand the expression pattern and regulation of uterine CaBP-9k gene, the expression of CaBP-9k mRNA and its regulation by estrogen (E2) and progesterone (P4) were investigated in the mouse uterus during late pregnancy (from day 12 to 18) and lactation. The expression levels of uterine CaBP-9k, estrogen receptor α (ERα) and progesterone receptor (PR) mRNAs were measured by Northern blot analysis. The expression levels of mouse uterine CaBP-9k mRNA gradually increased from pregnancy day 16 (P16), peaked at P18 (6.0-fold vs. P12) and declined at birth and during lactation. The expression levels of ERα and PR mRNAs indicated a similar fluctuation as CaBP-9k mRNA, suggesting the role of sex steroids/receptors in the regulation of CaBP-9k gene. To investigate effect of steroid hormone on CaBP-9k mRNA expression, three groups of animals were injected (s.c) with steroid hormone antagonists (RU486, tamoxifen, and ICI182780), respectively. RU486, a P4 antagonist, induced a significant decrease in CaBP-9k mRNA expression at 48 (3.2-fold) and 72 h (3.8-fold). However, tamoxifen and ICI182780, E2 antagonists, had no effect on CaBP-9k mRNA expression. Combined treatment with RU486 and ICI182780 did not further decrease the expression level of CaBP-9k mRNA when compared with RU486 treatment at 48 and 72 h. In addition, the treatment with RU40555, a glucocorticoid/progesterone antagonist, resulted in a decrease at 48 and 72 h following treatment. These results indicate that E2 is not likely involved in the regulation of CaBP-9k gene in the mouse uterus during late pregnancy and lactation. In conclusion, the present results suggest that P4, not E2 is a key regulator of CaBP-9k mRNA expression during late pregnancy and lactation.

The essential oils of Chamaecyparis obtusa promote hair growth through the induction of vascular endothelial growth factor gene.

Chamaecyparis obtusa (C. obtusa) is a conifer in the cypress family Cupressaceae, native to northeast Asia. The essential oils of C. obtusa have antibacterial and antifungal effects and several products such as hygienic bands, aromatics, and shampoos contain these oils as a natural source of antimicrobial/antifungal agents. Interestingly, some consumers suffering from baldness and/or other forms of hair loss have reported a hair growth promoting effect of shampoos containing these oils. In the present study, the hair growth promoting effect of C. obtusa oils was elucidated in an animal model. C. obtusa oils promoted the early phase of hair growth in shaved mice. In addition, we examined the molecular effect of C. obtusa oils on the regulation of hair morphogenesis and hair growth using the human keratinocyte cell line HaCaT. In the current study of hair growth regulating genes, the expressions of vascular endothelial growth factor (VEGF), transforming growth factor (TGF beta 1), and keratinocyte growth factor(KGF) have been analyzed by real-time PCR in HaCaT cells. The essential oils of C. obtusa were divided into seven fractions for treatment of HaCaT cells. VEGF transcripts were induced by fractions 6 and 7; however, TGF beta 1 and KGF mRNA levels were unchanged by C. obtusa oils or fractions. Fraction 7 was separated into seven sub-fractions and studied further. Sub-fractions E and D significantly increased VEGF and KGF gene expression without up-regulating the hair growth inhibition factor, TGF beta 1. The components of the two sub-fractions were further analyzed by gas chromatography and mass spectrometry. Cuminol, eucarvone, and calamenene were common to these two sub-fractions, although the effects of these individual components were not determined. Taken together, these results suggest that C. obtusa oils promote hair growth in an animal model and a positive regulator of hair growth, VEGF, was induced by particular components of these oils.

Complex regulation of Calbindin-D9k in the mouse placenta and extra-embryonic membrane during mid- and late pregnancy

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein mainly expressed in the duodenum, uterus and placenta, however, the role of CaBP-9k in the regulation of fetal growth remains to be elucidated. The present study was performed to investigate the expression pattern and regulation of CaBP-9k by antagonists of steroid hormones related with steroid hormone receptors during mid- and late pregnancy in mouse placenta and extra-embryonic membrane. The expression level of CaBP-9k increased in the placenta, while it decreased in the extra-embryonic membrane during pregnancy. The mRNA expression levels of estrogen receptor alpha (ERalpha) and progesterone receptor (PR) appeared to increase in both placenta and extra-embryonic membrane during pregnancy, suggesting that the ER and PR mRNA and protein expressions of placental CaBP-9k are positively correlated, but expressions of extra-embryonic membrane CaBP-9k are reversely correlated with ERalpha and PR mRNA levels. In addition, the present study indicates that the expressions of CaBP-9k mRNA and protein are differentially up- or down-regulated by antagonists of estrogen (E2) and progesterone (P4) in mouse placenta and extra-embryonic membranes, which suggests that E2 and P4 may be dominant factors in the regulation of the CaBP-9k. In particular, RU486, an antagonist of P4, down-regulated the mRNA and protein levels of placental CaBP-9k, whereas it up-regulated the protein level of extra-embryonic membrane CaBP-9k. In conclusion, we demonstrated that the CaBP-9k is distinctly regulated in the mouse placenta and extra-embryonic membrane, probably via sex steroid hormones (E2 and P4) and their receptors through a complex pathway. Extended studies are needed to verify relevant factors to regulate CaBP-9k gene and to provide further insight into roles of CaBP-9k gene in these tissues for the control of reproductive functions.

In vitro exposure to xenoestrogens induces growth hormone transcription and release via estrogen receptor-dependent pathways in rat pituitary GH3 cells

In this study, we employed an in vitro model to examine the effects of endocrine disruptors (EDs) in the regulation of growth hormone (GH) gene, an important hormone in growth, development and body composition. The rat pituitary cells, GH3, were treated with alkyl-phenols (APs), i.e., 4-tert-octyl-phenol (OP), p-nonyl-phenol (NP) or bisphenol A (BPA) for 24h in a dose-dependent manner (10(-5), 10(-6) and 10(-7)M) and in a time-dependent fashion (1, 3, 6, 12 and 24h) at a high concentration (10(-5)M). An anti-estrogen, ICI 182,780, was used to examine the potential involvement of estrogen receptor (ER) in the induction of GH by EDs through an ER-mediated pathway. Treatment with OP, NP and BPA induced a significant increase in GH gene expression at high and medium doses at 24h. ED-exposure induced a marked increase in GH gene transcription as early as 6h and peaked at 12h. Co-treatment with ICI 182,780 significantly attenuated ED-induced GH expression in GH3 cells. Interestingly, the level of in vitro GH release was significantly increased at 24h in response to OP, NP or BPA, whereas co-treatment with ICI 182,780 significantly reversed ED-induced GH secretion, indicating that ER may take part in both GH gene transcription and its release in these cells. In addition, the activation of extracellular signal-regulated kinases (ERKs), protein kinases B (Akt) or G protein in response to OP, NP or BPA at 24h was observed in this study. Exposure to these APs resulted in a rapid and significant activation of ERK phosphorylation, reflecting that EDs-induced response may involve both genomic and non-genomic pathways in these cells. Taken together, these results may provide new insight into the mode of ED-induced action in GH gene regulation as well as the biological pathway underlying these molecular events.

Dexamethasone differentially regulates renal and duodenal calcium-processing genes in calbindin-D9k and -D28k knockout mice

Glucocorticoids (GCs) appear to downregulate active calcium‐transporting genes in the duodenum, resulting in GC‐induced calcium‐absorbing disorder. In this study, we examined the effects of GCs on calcium‐processing genes in the duodenum and kidney and the compensatory mechanism in calbindin‐D9k (CaBP‐9k) and calbindin‐D28k (CaBP‐28k) knockout (KO) mice. In the duodenum, we observed compensatory increases in transient receptor potential vanilloid 6 (TRPV6) mRNAs in both calbindin KO mice and CaBP‐9k transcripts in CaBP‐28k KO mice, and their expressions were decreased by addition of a synthetic GC, dexamethasone (Dex, 10 mg kg−1). In addition, the expression of plasma membrane calcium ATPase 1b (PMCA1b) underwent a compensatory increase in CaBP‐9k KO mice, and was blocked by Dex, while the mRNA level of duodenal sodium‐calcium exchanger 1 was not altered by KO status or Dex. The renal transcriptional levels of TRPV5 in CaBP‐9k KO and CaBP‐9k in CaBP‐28k KO mice were upregulated in a compensatory manner, while the TRPV6 gene was downregulated following treatment with Dex in the kidney of CaBP‐28k KO mice. The immunological location of these duodenal proteins as a primary target of Dex‐involved regulation was not altered by Dex or KO status. To elucidate potential mechanism(s) of Dex‐induced compensatory gene expression, the levels of GC receptor (GR), vitamin D receptor (VDR) and parathyroid hormone receptor (PTHR) mRNA was also measured in these tissues. Duodenal VDR transcripts were induced in a compensatory manner in both types of KO mice, and were decreased by Dex. In addition, serum corticosterone levels in both KO mice were lower than in wild‐type mice. In conclusion, these results suggest that duodenal TRPV6 and CaBP‐9k genes appear to be a primary target for GC‐induced calcium‐absorbing disorder, through direct regulation of duodenal VDR transcription.

Uterine and placental expression of TRPV6 gene is regulated via progesterone receptor- or estrogen receptor-mediated pathways during pregnancy in rodents.

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an epithelial Ca2+ channel protein expressed in calcium absorbing organs. In the present study, we investigated the expression and regulation of uterine and placental TRPV6 during gestation in rodents. Uterine TRPV6 peaked at pregnancy day (P) 0.5, P5.5 and, P13.5 and was detected in uterine epithelium and glands of rats, while placental TRPV6 mRNA levels increased in mid-gestation. Uterine and placental TRPV6 mRNA levels in rats appear to cyclically change during pregnancy, suggesting that TRPV6 may participate in the implantation process. In addition, uterine TRPV6 mRNA is only expressed in placenta-unattached areas of the uterus, and uterine TRPV6 immunoreactivity was observed in luminal and glandular epithelial cells. In the placenta, TRPV6 was detected in the labyrinth and spongy zone. These results may indicate that TRPV6 has at least two functions: implantation of the embryo and maintenance of pregnancy. To investigate the pathway(s) mediating TRPV6 expression in rodents, anti-steroid hormone antagonists were injected prior to maximal TRPV6 expression. In rats, TRPV6 expression was reduced by RU486 (an anti-progesterone) through progesterone receptors, and ICI 182,780 (an anti-estrogen) blocked TRPV6 expression via estrogen receptors in mice. The juxtaposition of uterine and placental TRPV6 expressed in these tissues supports the notion that TRPV6 participates in transferring calcium ions between the maternal and fetal compartments. Taken together, TRPV6 gene may function as a key element in controlling calcium transport in the uterus between the embryo and the placenta during pregnancy.

Estrogen regulates the localization and expression of calbindin-D9k in the pituitary gland of immature male rats via the ERα-pathway

Estrogen (E2; estradiol) plays a key role in the regulation of many pituitary hormones. It exerts its effects by binding to the intracellular estrogen receptor (ER), which then functions as a transcription factor. Although E2 has been shown to regulate calbindin-D(9k) (CaBP-9k) in the female reproductive system of rodents, the effects of E2 on the regulation of CaBP-9k in male rats remain to be elucidated. To investigate E2-induced regulation of the pituitary CaBP-9k gene, immature male rats were injected with E2 daily for 3 consecutive days with a dose of 40 microg/kg body weight (BW). The expression levels of CaBP-9k mRNA and protein were analyzed by RT-PCR and Western blot analysis, respectively, in the absence and presence of ICI 182,780 (ICI), an E2 antagonist. In addition, the tissue localization of CaBP-9k was determined by immunohistochemistry. CaBP-9k was localized in the cytoplasm of a specific cell type (acidophils) in the anterior lobe of the pituitary gland and highly expressed in the intermediate lobe. Exposure to E2 increased the number of cells that stained positive for CaBP-9k. To determine which ER subtype is involved in CaBP-9k regulation in the pituitary, the immature rats were treated with propyl pyrazole triol (PPT, an ERalpha-selective ligand) or diarylpropionitrile (DPN, an ERbeta-selective ligand) for 3 days. Pituitary CaBP-9k expression was mainly mediated by PPT in immature male rats, whereas no significant alteration of pituitary CaBP-9k gene expression was observed after DPN treatment. In addition, the estrogenicity of PPT in the induction of CaBP-9k expression was completely blocked by an estrogen antagonist, ICI, indicating that pituitary CaBP-9k expression is solely induced by ERalpha. Taken together, these results suggest that pituitary CaBP-9k is induced by E2 in male rats and its expression is predominantly regulated by ERalpha, but not ERbeta.

The negative effect of dexamethasone on calcium-processing gene expressions is associated with a glucocorticoid-induced calcium-absorbing disorder

AIMSnAlthough dexamethasone (Dex) is used widely as an anti-inflammatory and immunosuppressive drug, Dex appears to have severe side-effects, including osteoporosis. This study determined the effects of Dex on duodenal and renal expressions of the calcium-processing genes transient receptor potential cation channel, subfamily V, member 5/6 (TRPV5/6), calbindin-D9k/-D28k (CaBP-9k/28k), Na+/Ca2+ exchanger 1 (NCX1), and plasma membrane Ca(2+)-ATPase (PMCA) 1b.nnnMAIN METHODSnMice were injected subcutaneously with Dex for 1 or 5 days. The mRNA and protein expression levels of these calcium-processing genes were measured by real-time PCR and immunohistochemistry/immunoblot analysis, respectively. In addition, serum parathyroid hormone (PTH) levels were measured following Dex treatments.nnnKEY FINDINGSnTreatment with Dex for 24 h resulted in the inductions of duodenal TRPV6, CaBP-9k and PMCA1b transcripts and renal TRPV5, CaBP-9k, and NCX1 transcripts, while it reduced the transcription of renal TRPV6. Although the expressional changes were weak, duodenal expressions of glucocorticoid receptor (GR), the vitamin D receptor (VDR), and renal expressions of the parathyroid hormone receptor (PTHR) and VDR were increased following 24 h treatment with Dex. A five-day treatment with Dex reduced the transcriptional levels of duodenal TRPV6 and CaBP-9k by 60%. Transcripts for VDR and GR in the duodenum increased marginally.nnnSIGNIFICANCEnThese results suggest that the expressions of TRPV6 and CaBP-9k in the duodenum appear to be a major regulatory target for glucocorticoids (GCs), and may be involved in the negative regulation of calcium absorption in GC-induced osteoporosis (GIO). The transcriptional regulation of TRPV6 and CaBP-9k in the duodenum seems complex given that there is an increase at 1-day treatment followed by a decrease at 5-day treatment.