Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH) 2 vitamin D [1,25(OH)2D3] action on calcium homeostasis. We assessed the rate of serum 45 Ca accumulation within 10 min of oral gavage in two strains of VDR-knockout (KO) mice (Leuven and Tokyo KO) and observed a 3-fold lower area under the curve in both KO strains. Moreover, we evaluated the expression of intestinal candidate genes involved in transcellular calcium transport. The calcium transport protein1 (CaT1) was more abundantly expressed at mRNA level than the epithelial calcium channel (ECaC) in duodenum, but both were considerably reduced (CaT1>90%, ECaC>60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D 9K expression was decreased only in the Tokyo KO, whereas plasma membrane calcium ATPase (PMCA 1b) expression was normal in both VDR-KOs. In Leuven wild-type mice, a high calcium diet inhibited (>90%) and 1,25(OH) 2D3 injection or low calcium diet induced (6-fold) duodenal CaT1 expression and, to a lesser degree, ECaC and calbindin-D 9K expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D 9K and PMCA1b expression, whereas CaT1 and ECaC expression remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular CaT1) is strongly vitamin D-dependent, and that calcium influx, probably interacting with calbindin-D 9K, should be considered as a rate-limiting step in the process of vitamin D-dependent active calcium absorption.

ABSTRACT1␣,25(OH) 2 -vitamin D strongly regulates the expression of the epithelial calcium channel CaT1. CaT1 expression is reduced in ERKO␣ mice and induced by estrogen treatment, pregnancy, or lactation in VDR WT and KO mice. Estrogens and vitamin D are thus independent potent regulators of the expression of this calcium influx mechanism, which is involved in active intestinal calcium absorption.Introduction: Active duodenal calcium absorption consists of three major steps: calcium influx into, transfer through, and extrusion out of the enterocyte. These steps are carried out by the calcium transport protein 1 (CaT1), calbindin-D 9K , and the plasma membrane calcium ATPase (PMCA 1b ), respectively. We investigated whether estrogens or hormonal changes during the female reproductive cycle influence the expression of these genes, and if so, whether these effects are vitamin D-vitamin D receptor (VDR) dependent. Materials and Methods:We evaluated duodenal expression patterns in estrogen receptor (ER)␣ and -␤ knockout (KO) mice, as well as in ovariectomized, estrogen-treated, pregnant, and lactating VDR wild-type (WT) and VDR KO mice. Results: Expression of calcium transporter genes was not altered in ERKO␤ mice. CaT1 mRNA expression was reduced by 55% in ERKO␣ mice, while the two other calcium transporter genes were not affected. Ovariectomy caused no change in duodenal expression pattern of VDR WT and KO mice, whereas treatment with a pharmacologic dose of estrogens induced CaT1 mRNA expression in VDR WT (4-fold) and KO (8-fold) mice. Pregnancy enhanced CaT1 expression equally in VDR WT and KO mice (12-fold). Calbindin-D 9K and PMCA 1b expression increased to a lesser extent and solely in pregnant VDR WT animals. In lactating VDR WT and KO mice, CaT1 mRNA expression increased 13 times, which was associated with a smaller increase in calbindin-D 9K protein content and PMCA 1b mRNA expression. Conclusions: Estrogens or hormonal changes during pregnancy or lactation have distinct, vitamin D-independent effects at the genomic level on active duodenal calcium absorption mechanisms, mainly through a major upregulation of the calcium influx channel CaT1. The estrogen effects seem to be mediated solely by ER␣.

Hyperglycemia is a common feature of the critically ill in general and of patients with sepsis in particular. Even a moderate degree of hyperglycemia appears detrimental for the outcome of critically ill patients, since maintenance of normoglycemia (blood glucose levels

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The translation of stem cell-based regenerative solutions from the laboratory to the clinic is often hindered by the culture conditions used to expand cell populations. Although fetal bovine serum (FBS) is widely used, regulatory bodies and safety concerns encourage alternative, xeno-free culturing practices. In an attempt to apply this approach to a bone-forming combination product of human periosteal progenitors (human periosteum derived cells) on a clinically used calcium phosphate carrier, FBS was substituted for human allogeneic serum (hAS) during cell expansion. It was found that cell proliferation was increased in hAS along with an apparent commitment to the osteogenic lineage, indicated by enhanced Runx2 expression, as well as alkaline phosphatase activity and matrix mineralization. Following analysis of signaling pathways, it was found that interferon-mediated signaling was downregulated, whereas JAK-STAT signaling was upregulated. STAT3 phosphorylation was enhanced in hAS-cultured human periosteum derived cells, inhibition of which ablated the proliferative effect of hAS. Furthermore, following in vivo implantation of hAS-cultured cells on NuOss scaffolds, enhanced bone formation was observed compared with FBS (71% increase, p < .001). Interestingly, the de novo-formed bone appeared to have a higher ratio of immature regions to mature regions, indicating that after 8 weeks implantation, tissue-formation processes were continuing. Integration of the implant with the environment appeared to be altered, with a decrease in calcium phosphate grain size and surface area, indicative of accelerated resorption. This study highlights the advantages of using humanized culture conditions for the expansion of human periosteal progenitors intended for bone regeneration.