Vitamin D biology revealed through the study of knockout and transgenic mouse models.

Early studies identifying vitamin D as an antirachitic factor led to studies in vitamin D-deficient models that resulted in a basic understanding of the mechanism of action of vitamin D. Recent studies using genetically modified mice have provided important new insight into the physiological role of vitamin D at target tissues and the functional significance of vitamin D target proteins, as well as the functional significance of proteins involved in the transport and metabolism of vitamin D. Studies using these mice have played an increasingly important role in elucidating the mechanisms involved in the control of calcium homeostasis and have provided evidence for a role of vitamin D in extraskeletal health.

Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k.

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

Vitamin D: molecular mechanism of action.

Vitamin D maintains calcium homeostasis and is required for bone development and maintenance. Recent evidence has indicated an interrelationship between vitamin D and health beyond bone, including effects on cell proliferation and on the immune system. New developments in our lab related to the function and regulation of target proteins have provided novel insights into the mechanisms of vitamin D action. Studies in our lab have shown that the calcium-binding protein, calbindin, which has been reported to be a facilitator of calcium diffusion, also has an important role in protecting against apoptotic cell death in different tissues including protection against cytokine destruction of osteoblastic and pancreatic beta cells. These findings have important implications for the therapeutic intervention of many disorders including diabetes and osteoporosis. Recent studies in our laboratory of intestinal calcium absorption using calbindin-D(9k) null mutant mice as well as mice lacking the 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inducible epithelial calcium channel, TRPV6, provide evidence for the first time of calbindin-D(9k) and TRPV6 independent regulation of active calcium absorption. Besides calbindin, the other major target of 1,25(OH)(2)D(3) in intestine and kidney is 25(OH)D(3) 24 hydroxylase (24(OH)ase), which is involved in the catabolism of 1,25(OH)(2)D(3). In our laboratory we have identified various factors that cooperate with the vitamin D receptor in regulating 24(OH)ase expression including C/EBP beta, SWI/SNF (complexes that remodel chromatin using the energy of ATP hydrolysis) and the methyltransferases, CARM1 and G9a. Evidence is also presented for C/EBP beta as a nuclear coupling factor that coordinates regulation of osteopontin by 1,25(OH)(2)D(3) and PTH. Our findings define novel mechanisms that may be of fundamental importance in understanding how 1,25(OH)(2)D(3) mediates its multiple biological effects.

Transgenic Expression of the Vitamin D Receptor Restricted to the Ileum, Cecum, and Colon of Vitamin D Receptor Knockout Mice Rescues Vitamin D Receptor-Dependent Rickets.

Although the intestine plays the major role in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action on calcium homeostasis, the mechanisms involved remain incompletely understood. The established model of 1,25(OH)2D3-regulated intestinal calcium absorption postulates a critical role for the duodenum. However, the distal intestine is where 70% to 80% of ingested calcium is absorbed. To test directly the role of 1,25(OH)2D3 and the vitamin D receptor (VDR) in the distal intestine, three independent knockout (KO)/transgenic (TG) lines expressing VDR exclusively in the ileum, cecum, and colon were generated by breeding VDR KO mice with TG mice expressing human VDR (hVDR) under the control of the 9.5-kb caudal type homeobox 2 promoter. Mice from one TG line (KO/TG3) showed low VDR expression in the distal intestine (<50% of the levels observed in KO/TG1, KO/TG2, and wild-type mice). In the KO/TG mice, hVDR was not expressed in the duodenum, jejunum, kidney, or other tissues. Growth arrest, elevated parathyroid hormone level, and hypocalcemia of the VDR KO mice were prevented in mice from KO/TG lines 1 and 2. Microcomputed tomography analysis revealed that the expression of hVDR in the distal intestine of KO/TG1 and KO/TG2 mice rescued the bone defects associated with systemic VDR deficiency, including growth plate abnormalities and altered trabecular and cortical parameters. KO/TG3 mice showed rickets, but less severely than VDR KO mice. These findings show that expression of VDR exclusively in the distal intestine can prevent abnormalities in calcium homeostasis and bone mineralization associated with systemic VDR deficiency.

Multi-Center Randomized Controlled Trial on the Effect of Triclosan-Coated Sutures on Surgical Site Infection after Colorectal Surgery.

BACKGROUND: Surgical site infection (SSI) remains the most frequent complication after colorectal resection. The role of sutures coated with antimicrobial agents such as triclosan in reducing SSI is controversial. METHODS: This was a multi-center randomized controlled trial with patients and outcome assessors blinded to treatment. The study was performed in four university referral hospitals. Patient candidates for elective colorectal resection were assigned randomly to abdominal incision closure with polyglactin 910 triclosan-coated sutures (triclosan group) or with polyglactin 910 without triclosan (control group). The primary outcome was the rate of SSI within 30 d after hospital discharge. The secondary outcomes were the overall rate of incision complications and length of hospital stay (LOS). RESULTS: Two hundred eighty-one patients (triclosan group: 140; control group: 141) were analyzed after randomization. The rate of SSI was 12.9% (18/140) in the triclosan group versus 10.6% (15/141) in the control group (odds ratio: 1.24; 95% confidence interval: 0.60-2.57; p=0.564). Secondary outcome analysis showed an overall incision complication rate of 38.3% in the control group versus 45.7% in the triclosan group (odds ratio: 1.36; 95% confidence interval: 0.84-2.18; p=0.208). Median LOS was 11 d in both groups (p=0.55). CONCLUSIONS: Surgical sutures coated with triclosan do not appear to be effective in reducing the rate of SSI.

Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin-D28k.

UNLABELLED: This study show for the first time that calbindin-D28k can prevent glucocorticoid-induced bone cell death. The anti-apoptotic effect of calbindin-D28k involves inhibition of glucocorticoid induced caspase 3 activation as well as ERK activation. INTRODUCTION: Recent studies have indicated that deleterious effects of glucocorticoids on bone involve increased apoptosis of osteocytes and osteoblasts. Because the calcium-binding protein calbindin-D28k has been reported to be anti-apoptotic in different cell types and in response to a variety of insults, we investigated whether calbindin-D28k could protect against glucocorticoid-induced cell death in bone cells. MATERIALS AND METHODS: Apoptosis was induced by addition of dexamethasone (dex; 10-6 M) for 6 h to MLO-Y4 osteocytic cells as well as to osteoblastic cells. Apoptosis percentage was determined by examining the nuclear morphology of transfected cells. Caspase 3 activity was evaluated in bone cells and in vitro. SELDI mass spectrometry (MS) was used to examine calbindin-D28k-caspase 3 interaction. Phosphorylation of calbindin-D28k was examined by 32P incorporation as well as by MALDI-TOF MS. ERK activation was determined by Western blot. RESULTS: The pro-apoptotic effect of dex in MLO-Y4 cells was completely inhibited in cells transfected with calbindin-D28k cDNA (5.6% apoptosis in calbindin-D28k transfected cells compared with 16.2% apoptosis in vector-transfected cells, p < 0.05). Similar results were observed in osteoblastic cells. We found that dex-induced apoptosis in bone cells was accompanied by an increase in caspase 3 activity. This increase in caspase 3 activity was inhibited in the presence of calbindin-D28k. In vitro assays indicated a concentration-dependent inhibition of caspase 3 by calbindin-D28k (Ki = 0.22 microM). Calbindin-D28k was found to inhibit caspase 3 specifically because the activity of other caspases was unaffected by calbindin-D28k. The anti-apoptotic effect of calbindin-D28k in response to dex was also reproducibly associated with an increase in the phosphorylation of ERK 1 and 2, suggesting that calbindin-D28k affects more than one signal in the glucocorticoid-induced apoptotic pathway. CONCLUSION: Calbindin-D28k, a natural non-oncogenic protein, could be an important target in the therapeutic intervention of glucocorticoid-induced osteoporosis.

Calcium transporter 1 and epithelial calcium channel messenger ribonucleic acid are differentially regulated by 1,25 dihydroxyvitamin D3 in the intestine and kidney of mice.

We examined the expression of calcium transporter 1 (CaT1) and epithelial calcium channel (ECaC) mRNA in the duodenum and kidney of mice. Intestinal CaT1 mRNA level increased 30-fold at weaning, coincident with the induction of calbindin-D(9k) expression. In contrast, renal CaT1 and ECaC mRNA expression was equal until weaning when ECaC mRNA is induced and CaT1 mRNA levels fall 70%. Long- and short-term adaptation to changes in dietary calcium (Ca) level and 1,25 dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] injection strongly regulated duodenal calbindin D(9k) and CaT1 mRNA. Following a single dose of 1,25(OH)(2)D(3), induction of CaT1 mRNA occurred rapidly (within 3 h, peak at 6 h of 9.6 +/- 0.8-fold) and preceded the induction of intestinal Ca absorption (significantly increased at 6 h, peak at 9 h). Neither renal CaT1 nor ECaC mRNA were strongly regulated by dietary calcium level or 1,25(OH)(2)D(3) injection. Our data indicate that CaT1 and ECaC mRNA levels are differentially regulated by 1,25(OH)(2)D(3) in kidney and intestine and that there may be a specialized role for CaT1 in kidney in fetal and neonatal development. The rapid induction of intestinal CaT1 mRNA expression by 1,25(OH)(2)D(3), and the marked induction at weaning, suggest that CaT1 is critical for 1,25(OH)(2)D(3)-mediated intestinal Ca absorption.

Incarcerated hernia in a trocar site following laparoscopic gastric bypass.

This paper reports a case of small bowel occlusion due to the herniation of an ileal ansa at the site of the insertion of a 12-mm trocar. A 28-year-old obese female patient underwent laparoscopic surgery for gastric bypass, the skin incisions of the trocar insertion sites were closed with absorbable sutures. Small bowel occlusion occurred on post-operative day 6 and the patient again underwent laparoscopic surgery. Laparoscopic exploration confirmed the suspected presence of the incarcerated hernia of an ileal ansa at the site of the trocar insertion which was freed without any need for bowel resection. The fascial defect was successfully closed under direct vision with the use of a special system of fascial sutures.

Vitamin D and intestinal calcium absorption.

The principal function of vitamin D in calcium homeostasis is to increase calcium absorption from the intestine. Calcium is absorbed by both an active transcellular pathway, which is energy dependent, and by a passive paracellular pathway through tight junctions. 1,25Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) the hormonally active form of vitamin D, through its genomic actions, is the major stimulator of active intestinal calcium absorption which involves calcium influx, translocation of calcium through the interior of the enterocyte and basolateral extrusion of calcium by the intestinal plasma membrane pump. This article reviews recent studies that have challenged the traditional model of vitamin D mediated transcellular calcium absorption and the crucial role of specific calcium transport proteins in intestinal calcium absorption. There is also increasing evidence that 1,25(OH)(2)D(3) can enhance paracellular calcium diffusion. The influence of estrogen, prolactin, glucocorticoids and aging on intestinal calcium absorption and the role of the distal intestine in vitamin D mediated intestinal calcium absorption are also discussed.

New insights into the mechanisms of vitamin D action.

The biologically active metabolite of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a secosteroid whose genomic mechanism of action is similar to that of other steroid hormones and is mediated by stereospecific interaction of 1,25(OH)(2)D(3) with the vitamin D receptor (VDR) which heterodimerizes with the retinoid X receptor (RXR). After interaction with the vitamin D response element (VDRE) in the promoter of target genes, transcription proceeds through the interaction of VDR with coactivators and with the transcription machinery. The identification of the steps involved in this process has been a major focus of recent research in the field. However, the functional significance of target proteins as well as the functional significance of proteins involved in the transport and metabolism of vitamin D is also of major importance. Within the past few years much new information has been obtained from studies using knockout and transgenic mice. New insight has been obtained using this technology related to the physiological significance of the vitamin D binding protein (DBP), used to transport vitamin D metabolites, as well as the physiological significance of target proteins including 25-hydroxyvitamin D(3) 24-hydroxylase (24(OH)ase), 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (1 alpha-(OH)ase), VDR, and osteopontin. The crystal structure of the DBP and the ligand binding domain of the VDR have recently been reported, explaining, in part, the unique properties of these proteins. In addition novel 1,25(OH)(2)D(3) target genes have been identified including the epithelial calcium channel, present in the proximal intestine and in the distal nephron. Thus in recent years a number of exciting discoveries have been made that have enhanced our understanding of mechanisms involved in the pleiotropic actions of 1,25(OH)(2)D(3).