Myosin 1a Regulates Osteoblast Differentiation Independent of Intestinal Calcium Transport

Myosin 1A (Myo1a) is a mechanoenzyme previously thought to be located exclusively in the intestinal epithelium. It is the principle calmodulin-binding protein of the brush border. Based on earlier studies in chickens, we hypothesized that Myo1a facilitates calcium transport across the brush border membrane of the intestinal epithelium, perhaps in association with the calcium channel Trpv6. Working with C2Bbe1 cells, a human intestinal epithelial cell line, we observed that overexpression of Myo1a increased, whereas the antisense construct blocked calcium transport. To further test this hypothesis, we examined mice in which either or both Myo1a and Trpv6 had been deleted. Although the Trpv6-null mice had decreased intestinal calcium transport, the Myo1a-null mouse did not, disproving our original hypothesis, at least in mice. Expecting that a reduction in intestinal calcium transport would result in decreased bone, we examined the skeletons of these mice. To our surprise, we found no decrease in bone in the Trpv6-null mouse, but a substantial decrease in the Myo1a-null mouse. Double deletions were comparable to the Myo1a null. Moreover, Myo1a but not Trpv6 was expressed in osteoblasts. In vitro, the bone marrow stromal cells from the Myo1a-null mice showed normal numbers of colony-forming units but marked decrements in the formation of alkaline phosphatase–positive colonies and mineralized nodules. We conclude that Myo1a regulates osteoblast differentiation independent of its role, if any, in intestinal calcium transport, whereas Trpv6 functions primarily to promote intestinal calcium transport with little influence in osteoblast function.

Evidence for a Role of Prolactin in Calcium Homeostasis: Regulation of Intestinal Transient Receptor Potential Vanilloid Type 6, Intestinal Calcium Absorption, and the 25-Hydroxyvitamin D3 1α Hydroxylase Gene by Prolactin

Increased calcium transport has been observed in vitamin D-deficient pregnant and lactating rats, indicating that another factor besides 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is involved in intestinal calcium transport. To investigate prolactin as a hormone involved in calcium homeostasis, vitamin D-deficient male mice were injected with 1,25(OH)2D3, prolactin, or prolactin + 1,25(OH)2D3. Prolactin alone (1 μg/g body weight 48, 24, and 4 h before termination) significantly induced duodenal transient receptor potential vanilloid type 6 (TRPV6) mRNA (4-fold) but caused no change in calbindin-D9k. Combined treatment with 1,25(OH)2D3 and prolactin resulted in an enhancement of the 1,25(OH)2D3 induction of duodenal TRPV6 mRNA, calbindin-D9k mRNA, and an induction of duodenal calcium transport [P < 0.05 compared with 1,25(OH)2D3 alone]. Because lactation is associated with an increase in circulating 1,25(OH)2D3, experiments were done to determine whether prolactin also has a direct effect on induction of 25-hydroxyvitamin D3 1α hydroxylase [1α(OH)ase]. Using AOK B-50 cells cotransfected with the prolactin receptor and the mouse 1α(OH)ase promoter −1651/+22 cooperative effects between prolactin and signal transducer and activator of transcription 5 were observed in the regulation of 1α(OH)ase. In addition, in prolactin receptor transfected AOK B-50 cells, prolactin treatment (400 ng/ml) and signal transducer and activator of transcription 5 significantly induced 1α(OH)ase protein as determined by Western blot analysis. Thus, prolactin, by multiple mechanisms, including regulation of vitamin D metabolism, induction of TRPV6 mRNA, and cooperation with 1,25(OH)2D3 in induction of intestinal calcium transport genes and intestinal calcium transport, can act as an important modulator of vitamin D-regulated calcium homeostasis.