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"CALCIUM HOMEOSTASIS: REGULATORY AND COMPENSATORY MECHANISMS
INTESTINE, KIDNEY, BONE AND SKIN"

by
Dare V. Ajibade
MD/PhD Program
B.A. 2004, Rutgers University, New Brunswick



Thesis Advisor: Sylvia Christakos, Ph.D.
Professor
Department of Biochemistry& Molecular Biology

Tuesday, March 22, 2011
MSB E-609, 12:00 P.M.


Abstract

This three part thesis sought to increase the current understanding involving regulatory and compensatory mechanisms of calcium homeostasis. Firstly, the role of prolactin in calcium homeostasis was explored. 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 (1g/g body weight at 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 mRNA. 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 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 1 alpha hydroxylase (1(OH)ase). Using AOK B-50 cells co-transfected with the prolactin receptor and the mouse 1(OH)ase promoter -1651/+22, cooperative effects between prolactin and signal transducer and activator of transcription 5 (STAT5) were observed in the regulation of 1(OH)ase. In addition, in prolactin receptor transfected AOK B-50 cells, prolactin treatment (400 ng/ml) and STAT5 significantly induced 1(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.

The second part of this thesis examined skeletal integrity in mice lacking TRPV6 under restricted calcium condition. Active intestinal calcium absorption is important for maintaining normal calcium, especially when dietary calcium supply is low. Current models of active intestinal calcium absorption suggest TRPV6 aids in mediating calcium transfer across the intestinal apical membrane. Previous studies using TRPV6 null mutant (TRPV6KO) mice on low calcium diet demonstrated a less pronounced induction, but substantial intestinal calcium transport compared to wild type mice. However, the impaired intestinal calcium absorption did not lead to hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis. In this study, we report a detailed analysis of the bone phenotype of TRPV6KO mice compared to TRPV6WT mice fed a normal to high (~ 1%) or low (~ 0.02%) calcium diet. Bone morphology and remodeling was evaluated by pQCT, CT, histomorphometry and serum parameters. Restricted dietary calcium did not produce a genotype specific effect on bone perimeter examined except for increased osteoid surface. The amount of unmineralized bone mass, as suggested by osteiod surface, was increased in TRPV6KO compared to TRPV6WT mice under low dietary calcium condition. Taken together, these findings suggest impressive redundancy and compensatory mechanisms to maintain normacalcemia in the absence of TRPV6 gene. However, the lack of TRPV6 is suggestive of increase risk of osteomalacia as shown by hyperosteiodosis under restricted calcium condition.

The final part of this thesis suggests a regulatory role for Vitamin D and/or calcium in post-morphogenic hair follicle cycling. The skin has long been known as a target organ for vitamin D action. Although 1,25(OH)2D3 and calcium promote differentiation of keratinocytes, current literature does not suggest a role for 1,25(OH)2D3 and/or calcium in hair follicle development. Vitamin D receptor (VDR) deficient mice have been shown to develop alopecia and a role for unliganded VDR in hair follicle development through the WNT signaling (-catenin/Lef 1) pathway has been reported. Whether 1,25(OH)2D3 and/or calcium have a direct effect on hair follicle development is, as yet, unresolved. Herein, we report calbindin-D9k null mutant (KO) pups generated from calbindin-D9k KO parents fed a vitamin D deficient, low calcium (0.4%) diet developed alopecia. The pups appear phenotypically normal until 13 days of age, after which the coat begins to look ruffled and progressively sheds in a caudocephalic direction. This telogenic effluvium results in a transient truncal alopecia totalis at 20-23 days of age, sparing only the head and a tuff around the base of the tail. The alopecia is complete by 23 days with a spontaneous recovery completed by the end of the 4th week. Wild type mice (C57BL6) developed a similar but inconsistent phenotype under identical protocol. Histological and immunohistochemistry (IHC) examination of whole skin thickness suggest deregulated follicle differentiation leading to development of utricles. The phenotype was partially rescued in pups from mothers feed a vitamin D deficient/ high Ca2+ (2%), high lactose (20%) diet during lactation, while complete rescue was seen in pups fostered to mothers on vitamin D (2,000 IU/D2/kg diet) and calcium (1%) in the diet. Taken together, for the first time, our result suggest a role of calcium in hair cycling where vitamin D and/ or calcium directly affect postmorphogenic hair follicle development and that deficiency of vitamin D and/or calcium leads to non-cicatricial alopecia.


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