Interaction of 1,25-Dihydroxyvitamin-D₃with Keratinocytes and Fibroblasts from Skin of Normal Subjects and a Subject with Vitamin-D-Dependent Rickets, Type II: A Model for Study of the Mode of Action of 1,25-Dihydroxyvitamin D₃*

Abstract

The binding of [ 3 H]l,25-dihydroxyvitamin D 3 (1,25-(OH) 2 -[ 3 H]D3) was examined in the cytosol of epidermal keratinocytes and dermal fibroblasts grown from normal human skin. Both cell types contained macromolecules with high affinity for 1,25-(OH) 2 D 3 , as demonstrated by sucrose density gradient centrifugation, saturation binding analysis, and DNAcellulose chromatography. Scatchard analysis of cytosol binding of the hormone yielded affinity constants of 1.0 x 10" 10 and 0.8 x 10" 10 M and binding capacities of 6.4 and 8.7 fmol/mg protein for fibroblasts and keratinocytes, respectively. In parallel studies, binding of 1,25-(OH) 2 -[ 3 H]D3 was evaluated in the cytosol from keratinocytes and fibroblasts cultured from a skin biopsy of a patient with vitamin D-dependent rickets, type II (DDR-II), an inheritable disorder characterized by extreme end-organ resistance to the action of 1,25-(OH)2D 3 . Multiple analyses by sucrose density gradient or saturation binding assays failed to reveal specific saturable binding of hormone, suggesting that in this disease the lack of an effective cytosolic receptor protein may account for end-organ insensitivity to the hormone. To evaluate the responsiveness of fibroblasts to 1,25-(OH) 2 D3, we studied the effect of 1,25-(OH) 2 D 3 on cell growth. 1,25-(OH) 2 D 3 (10~1 0 -10~6 M) caused a dose-dependent inhibition of cell growth in receptor-positive normal fibroblasts, whereas these doses of hormone did not affect the growth of receptor-defective DDR-II cells. Hence, the presence of an effective cytosolic receptor in normal skin fibroblasts may be necessary for expression of the growth inhibitory effect of 1,25-(OH) 2 D 3 . (J Clin Endocrinol Metab 56: 824, 1983) V ITAMIN D 3 is synthesized photochemically in the skin and then sequentially hydroxylated to 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ], the biologically active form of the vitamin (1). This hormone acts on target tissues, such as the intestine, after binding to a specific cytosolic receptor protein. Recently, receptor-like binding proteins for 1,25-(OH) 2 D 3 have been demonstrated in intact mammalian skin (2-5), raising the possibility that the hormone might function in this tissue. Subsequent studies by Eil and Marx (6) have shown that

Specific 1,25-Hydroxycholecalciferol Receptors and Stimulation of 25-Hydroxycholecalciferol- 24R Hydroxylase in Human Amniotic Cells

“…A single peak with binding activity is observed upon sucrose-gradient analysis of the hypertonic cytosolic extract. Its sedimentation coefficient is in agreement with those reported by others in either cultured skin fibroblasts (3,5) or in other target tissues (22,25). The specificity of this macromolecule for 1,25(OH)2D3 is evident as incubation of the extracts in presence of 100-fold excess of unlabeled 1 ,25(OH)2D3 completely abolishes the 3.0-3.6 S peak of radioactivity.…”

“…These receptors exhibit, under equilibrium conditions, Kd properties similar to those observed for cultured skin fibroblasts (7,21) and for other target tissues in different species (22,23). Despite differences in cell extract preparation and protein measurement methods among investigators, the estimated maximal binding capacities are of the same order of rnagnitupe as those reported by others for serially cultured'dermal fibroblasts (3,5,21). The number of binding sites varied widely from one cell strain to another.…”

“…All had a normal karyotype, five were male and one was female. Hypertonic cytosol extracts were prepared by sonication followed by centrifugation at Previous reports have shown that rat, murine, and human skin and cultured fibroblasts possessed an effector system for 1,25(OH)2D3 (1)(2)(3)(4)(5) suggesting that these tissues could serve as targets for the hormonal form of vitamin D3. Moreover, it has been established that these cells were responsive to 1 ,25(OH)2D3…”

Hereditary vitamin D resistant rickets caused by a novel mutation in the vitamin D receptor that results in decreased affinity for hormone and cellular hyporesponsiveness.

“…Hereditary 1,25-dihydroxyvitamin D resistant rickets (HVDRR), also known as vitamin D-dependent rickets, type II, is an autosomal recessive disorder that has been shown to be caused by mutations in the VDR (1,2). The disease is characterized by early onset rickets, hypocalcemia, secondary hyperparathyroidism, elevated 1,25(OH) 2 D 3 levels, and resistance to 1,25(OH) 2 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). In the receptor positive cases the VDR was shown to exhibit a decreased binding affinity for DNA.…”

Bone changes caused by experimental Solanum malacoxylon poisoning in rabbits

“…bone disease, psoriasis, and cancer (Clemens et al 1983, Rice et al 1992, Walters 1992, Bikle & Pillai 1993. Changes in cell differentiation induced by calcinogenic plants have been analysed in skin, aorta and lung , 2004, Portiansky et al 2002.…”