Yosef Weisman

1α,25-dihydroxyvitamin D3 (Calcitriol) inhibits hypoxia-inducible factor-1/vascular endothelial growth factor pathway in human cancer cells

In vitro and in vivo studies have shown that 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits angiogenesis in cancer. We now examined whether the antiangiogenic effects of 1,25(OH)2D3 are mediated by the hypoxia-inducible factor (HIF)-1 pathway. Our results showed that 1,25(OH)2D3 reduces the protein expression of both the regulated HIF-1α subunit and the vascular endothelial growth factor (VEGF) in various human cancer cells. 1,25(OH)2D3 also inhibited HIF-1 transcriptional activity (measured by reporter gene assay) as well as HIF-1 target genes, including VEGF, ET-1, and Glut-1. We also showed that 1,25(OH)2D3 inhibits cell proliferation under hypoxia. Using HIF-1α knockout colon cancer cells, we show that the inhibition of the hypoxia-induced VEGF by 1,25(OH)2D3 is mediated through a HIF-dependent pathway. Because HIF-1 is a major positive contributor in human tumorigenesis and angiogenesis, we believe that its inhibition by 1,25(OH)2D3 strengthens the rationale to use vitamin D and its low-calcemic analogues in cancer chemoprevention and therapy. [Mol Cancer Ther 2007;6(4):1433–9]

Long-term intracaval calcium infusion therapy in end-organ resistance to 1,25-dihydroxyvitamin D

Two boys aged six and four with the syndrome of hereditary resistance to 1,25-dihydroxyvitamin D3 with rickets alopecia and growth retardation are presented. After unsuccessful therapeutic trials with pharmacologic doses of vitamin D or its active metabolites, the patients were treated by long-term intracaval infusions of calcium through an implantable catheter. A total of 0.5 to 0.9 g of elemental calcium was infused daily for 18 months and the serum calcium concentration was maintained at 9 to 10 mg/dl. Bone pain subsided within one week of treatment. Serum phosphorus, immunoreactive parathyroid hormone, and 1,25-dihydroxyvitamin D concentrations and alkaline phosphatase activity were normalized within four to nine months. Radiographs of the knees and hands revealed progressive healing of rickets with complete resolution after one year of treatment. The patients gained 12 cm and 8 cm per year in height as compared with 3 cm and 2 cm, respectively, in the previous year. A transilial bone biopsy obtained from one patient prior to treatment revealed severe osteomalacia associated with osteitis fibrosa. A follow-up biopsy examined after 12 months of therapy showed almost complete healing of osteomalacia and normal mineralization. These observations indicate the following: (1) Long-term intracaval calcium infusions are an effective mode of therapy for these patients, and (2) When adequate serum calcium and phosphorus concentrations are maintained, healing of rickets and normal growth rate could be achieved even in the absence of a normal 1,25-dihydroxyvitamin D3 receptor-effector system.

Regulation of proliferation of rat cartilage and bone by sex steroid hormones.

We have demonstrated previously that 17 beta-estradiol (E2) stimulates proliferation of skeletal tissues, both in vivo and in vitro, as measured by increased DNA synthesis and creatine kinase (CK) specific activity. The effect of E2 on bone is sex specific. E2 is active only in females and androgens only in males. By contrast, in cartilage of both sexes, dihydrotestosterone (DHT) as well as E2 stimulates CK specific activity and DNA synthesis. In bone, we find that sex steroids stimulate skeletal cell proliferation in gonadectomized as well as in immature rats. Ovariectomized (OVX) rats, between 1 and 4 weeks after surgery, show stimulation of CK by E2. The basal activity and response of CK changes with the varying endogenous levels of E2 in cycling rats, in which the highest basal activity is at proestrus and estrus and the highest response is in diestrus. In rats of all ages tested, both the basal and stimulated specific activity of CK is higher in diaphysis and epiphysis than in the uterus, or in the adipose tissue adjacent to the uterus, which has a response similar to that of the uterus itself. The effect of E2 in vivo, and in chrondroblasts and osteoblasts in vitro, is inhibited by high levels of the antiestrogen tamoxifen which, by itself, in similar high concentrations, shows stimulatory effects. In addition to the sex steroids, skeletal cells are also stimulated by secosteroid and peptide calciotrophic hormones. The interactions of the sex steroids with these hormones modulate the response of cartilage and bone cells to both sex steroids and the other calciotrophic hormones. These results provide the first steps towards understanding the regulation of bone cell proliferation and growth by the concerted action of a variety of hormones and growth factors.

Maternal-perinatal interrelationships of vitamins D metabolism in rats

In pregnant rats it has been possible to show that the distribution of cholecalciferol metabolites in their fetuses reflects the distribution of these metabolites in the blood. In these experiments, pregnant rats were maintained on a vitamin D deficient diet but were supplemented with radiolabelled cholecalciferol. The metabolites found were 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol and, to a lesser extent, cholecalciferol. 1,25-Dihydroxycholecalciferol was not detected in fetal tissues, despite the ability of fetal kidney homogenates to hydroxylate 25-hydroxycholecalciferol in C-1. Kidney homogenates of newborn pups were found to possess marked activity of 25-hydroxycholecalciferol-24-hydroxylase, which was retained even in hypocalcemic pups born to pregnant rats that were fed a low-calcium diet. Injection of radiolabeled cholecalciferol to newborn pups resulted in the formation of 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. 1,25-Dihydroxycholecalciferol was not detected. Tissues thought of as target organs for vitamin D (in pregnant rats), namely, intestine, kidney and bone, were found to contain none or very little 1,25-dihydroxycholecalciferol. Mammary glands obtained from lactating rats were found to contain mainly the unchanged vitamin.

Hormonal stimulation of bone cell proliferation.

The recent demonstration of estrogen receptors in bone derived cells has stimulated the study of direct effects of sex steroids on bone. We have shown direct stimulation of proliferation by 17 beta-estradiol (E2) of ROS 17/2.8 rat osteogenic osteosarcoma cells, and other bone-derived cells in culture, as well as sex-specific stimulation of diaphyseal bone in vivo by estrogen and testosterone, using [3H]thymidine incorporation into DNA and stimulation of the specific activity of creatine kinase as markers. ROS 17/2.8 cells were used as models of osteoblast-like cells to study the reciprocal modulation of stimulation of bone cell proliferation by sequential treatment by sex steroid and calciotrophic hormones. Pretreatment with 1,25(OH)2D3 and PTH augmented stimulation by E2, while pretreatment with PGE2 followed by E2 resulted in no additional stimulation. Reciprocally, pretreatment with E2 significantly reduced the response to PGE2 while showing an insignificant effect on the response to the other hormones. Gonadectomized Wistar-derived rats provided a useful model system for study of postmenopausal osteoporosis. In diaphyseal bone, [3H]thymidine incorporation and creatine kinase activity decreased 4 weeks after gonadectomy. At that time, a single i.p. injection of E2 in females, and testosterone in males, resulted in a highly significant increase in both these parameters within 24 h.

Long-term physical exercise retards trabecular bone loss in lumbar vertebrae of aging female mice

SummaryThe present study examined the effect of long-term, moderate physical exercise on trabecular bone volume (TBV), calcium content,3H-proline uptake, and the activities of alkaline and acid phosphatases in lumbar vertebrae of aging and senescent mice. It became apparent that if physical activity starts at an early stage of life, i.e., prior to middle age and is extended until old age, it exerts beneficial effects on trabecular bone mass and mineralization. Such a positive effect is not obtained if the training program is initiated after middle age. The training-induced reduction in bone loss was accompanied by a significant decrease in acid phosphatase activity whereas no changes took place with regard to the activity of alkaline phosphatase. Long-term physical exercise also enhanced the uptake of3H-proline by lining cells along the bone trabecules. In spite of its moderate nature, the endured training program served as a stress factor for the involved animals, a fact that was manifested by an increase in the serum levels of corticosterone. Thus, it seems that whereas young animals respond favorably to such a stimulatory stress, older animals lose this ability of adaptation.

Pseudohypoparathyroidism type 1a presenting as congenital hypothyroidism

PSEUDOHYPOPARATHYROID1SM TYPE 1 is an inherited metabolic disorder characterized by hypocalcemia and hyperphosphatemia, which are caused by end organ resistance to the action of PTH?. 2 Most of these patients have, in addition, the skeletal abnormalities of Albright hereditary osteodystrophy. 2 The molecular basis for the resistance to PTH in most patients ( P H P l a ) is an impairment in c A M P synthesis 3 caused by deficient activity of guanine nucleotide regulatory protein of adenylate cyclase (Nprotein), 4-7 a plasma membrane protein that couples hormone receptors to the catalytic uni t of adenylate cyclase. 8 The localization of the defect distal to the hormone receptor implies that resistance to other hormones that act via c A M P may occur in PHP-1. Indeed, resistance to TSH, glucagon, ADH, and gonadotropins has been demonstrated in patients with PHP-1.6, 7 We describe a child in whom hypothyroidism was diagnosed shortly after birth; at the age of 5 years, a diagnosis of PHP-1 a was made.

Pseudohypoparathyroidism type Ia: two new heterozygous frameshift mutations in exons 5 and 10 of the Gsα gene

Pseudohypoparathyroidism type Ia (PHP-Ia) is a hereditary disease characterized by resistance to PTH and other hormones that act via cAMP. Patients have deficient activity of Gsα, the α subunit of the G protein, which couples hormone receptors to stimulation of adenylate cyclase. We describe two new mutations discovered in two sporadic patients with PHP-Ia. Using genomic DNA, we have amplified exons 2–13 of the Gsα gene (GNAS1) by PCR, and sequenced the resulting products. Both patients had Albrights hereditary osteodystrophy, resistance to multiple hormones, and deficient Gsα activity. In the first patient, a deletion of a C in exon 5 at codon 115 was found. In the second patient, an insertion of a C in exon 10 at codon 267 was detected. Both these heterozygous mutations cause frameshift, and predict decreased production of Gsα. This report adds two new Gsα mutations to the known ten mutations recently described.

Vitamin D metabolites in human milk

The concentrations of unconjugated 25-OHD, 24, 25(OH)2D, and 1,25(OH)2D were measured in human milk by competitive protein-binding radioassays following successive preparative Sephadex LH-20 chromatography and HPLC. The mean (+/- SE) concentration of 25-OHD was 0.37 +/- 0.03 ng/ml, of 24,25(OH)2D was 24.8 +/- 1.9 pg/ml, and of 1,25(OH)2D was 2.2 +/-0.1 pg/ml. The concentration of 25-OHD3 in milk as determined by HPLC and UV detection at 254 nm was 0.27 +/- 0.08 ng/ml. The milk concentrations of vitamin D metabolites did not correlate with the maternal serum 25-OHD levels. The total amounts of unconjugated vitamin D metabolites correspond to the known low bioassayable vitamin D antirachitic activity in human milk.

25 hydroxy-vitamin D3-1α hydroxylase expression and activity in cultured human osteoblasts and their modulation by parathyroid hormone, estrogenic compounds and dihydrotestosterone

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.