Interaction of 1,25-dihydroxyvitamin-D 3 with 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 D 3

Thomas Clemens, J. S. Adams, N. Horiuchi, B. A. Gilchrest, H. Cho, Y. Tsuchiya, N. Matsuo, T. Suda, M. F. Holick

Research output: Contribution to journalArticle

Abstract

The binding of [ 3H]1,25-dihydroxyvitamin D 3 (1,25-(OH) 2-[ 3H]D 3) was examined in the cytosol of epidermal keratinocytes and dermal fibroblasts grown from normal human skin. Both cell types contained macromolecues with high affinity for 1,25-(OH) 2D 3, as demonstrated by sucrose density gradient centrifugation, saturation binding analysis, and DNA-cellulose chromatography. Scatchard analysis of cytosol binding of the hormone yielded affinity constants of 1.0 x 10 -10 and 0.8 x 10 -10M 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-[ 3H]D 3 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) 2D 3, we studied the effect of 1,25-(OH) 2D 3 on cell growth. 1,25-(OH) 2D 3 (10 -10-10 -6M) 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) 2D 3.

Original languageEnglish (US)
Pages (from-to)824-830
Number of pages7
JournalJournal of Clinical Endocrinology and Metabolism
Volume56
Issue number4
StatePublished - 1983
Externally publishedYes

Fingerprint

Rickets
Fibroblasts
Keratinocytes
Vitamin D
Skin
Hormones
Cytosol
Cell growth
Growth
Sucrose
Density Gradient Centrifugation
Centrifugation
Biopsy
Chromatography
1,25-dihydroxyvitamin D
Assays
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology, Diabetes and Metabolism

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Interaction of 1,25-dihydroxyvitamin-D 3 with 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 D 3. / Clemens, Thomas; Adams, J. S.; Horiuchi, N.; Gilchrest, B. A.; Cho, H.; Tsuchiya, Y.; Matsuo, N.; Suda, T.; Holick, M. F.

In: Journal of Clinical Endocrinology and Metabolism, Vol. 56, No. 4, 1983, p. 824-830.

Research output: Contribution to journalArticle

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abstract = "The binding of [ 3H]1,25-dihydroxyvitamin D 3 (1,25-(OH) 2-[ 3H]D 3) was examined in the cytosol of epidermal keratinocytes and dermal fibroblasts grown from normal human skin. Both cell types contained macromolecues with high affinity for 1,25-(OH) 2D 3, as demonstrated by sucrose density gradient centrifugation, saturation binding analysis, and DNA-cellulose chromatography. Scatchard analysis of cytosol binding of the hormone yielded affinity constants of 1.0 x 10 -10 and 0.8 x 10 -10M 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-[ 3H]D 3 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) 2D 3, we studied the effect of 1,25-(OH) 2D 3 on cell growth. 1,25-(OH) 2D 3 (10 -10-10 -6M) 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) 2D 3.",
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AU - Clemens, Thomas

AU - Adams, J. S.

AU - Horiuchi, N.

AU - Gilchrest, B. A.

AU - Cho, H.

AU - Tsuchiya, Y.

AU - Matsuo, N.

AU - Suda, T.

AU - Holick, M. F.

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