Dalia Somjen

25-Hydroxyvitamin D3-1α-Hydroxylase Is Expressed in Human Vascular Smooth Muscle Cells and Is Upregulated by Parathyroid Hormone and Estrogenic Compounds

Background—1,25(OH)2 vitamin D3 exerts multiple effects in human vascular smooth muscle cells (VSMCs). We therefore tested the possibility that VSMCs possess an endogenous 25-hydroxyvitamin D3-1α-hydroxylase system, the final enzyme in the biosynthetic pathway of 1,25(OH)2D3. Methods and Results—We assessed the expression and activity of 25-hydroxyvitamin D3-1α-hydroxylase by real-time polymerase chain reaction and the conversion of 25(OH)D3 into 1,25(OH)2D3. First, 25-hydroxyvitamin D3-1α-hydroxylase mRNA was identified in cultured VSMCs by real-time polymerase chain reaction. Second, in cells treated daily (3 days) with parathyroid hormone (66 nmol/L), estradiol-17β (30 nmol/L), raloxifene (3 μmol/L), and the phytoestrogens genistein (3 μmol/L), biochainin A (3 μmol/L), and 6-carboxy biochainin A (30 nmol/L), 25-hydroxyvitamin D3-1α-hydroxylase mRNA increased by 43±13%, (P<0.05) 7±24% (P=NS), 176±28% (P<0.01), 65±11% (P<0.05), 152±24% (P<0.01), and 71±9% (P<0.05), respectively. Third, production of 1,25(OH)2D3 from 25(OH)D3 was seen with a Km of 25 ng/mL and increased dose dependently after treatment with parathyroid hormone, genistein, and the phytosetrogen derivative 6-carboxy biochainin A. Estradiol-17β and biochainin A also increased the generation of 1,25(OH)2D3 by 40±23% (P<0.05) and 55±13% (P<0.05), respectively. Conclusions—We provide here the first evidence for the expression of an enzymatically active 25(OH)D3-1α-hydroxylase system in human VSMCs, which can be upregulated by parathyroid hormone and estrogenic compounds. Because exogenous vitamin D inhibits VSMC proliferation, the role of this system as an autocrine mechanism to curb changes in VSMC proliferation and phenotype is a subject for future investigation.

Stimulation by oestradiol-17β of specific cytoplasmic and chromosomal protein synthesis in immature rat uterus

Abstract 1. 1) We have studied the early induction by oestradiol-17β, of the synthesis of specific proteins in the cytosol and chromatin fractions of the immature (19–21 days) rat uterus. Surviving uteri from rats given 5 ug of oestradiol-17β by intraperitoneal injection were incubated for l h with 3H-leucine; uteri from control rats were incubated with 14C-leucine. Corresponding fractions were pooled, and the 3H/14C ratio in electrophoretically separated protein components was determined. 2. 2) The oestradiol-17β-induced protein (IP) originally described by Gorski and Notides (1969) was found to be localized in the 106g supernatant fraction of uteri homogenized in a medium which preserves the integrity of nuclei (0.25 M sucrose, 50 mM Tris buffer pH 7.5, 25 mM KCl and 5 mM MgCl2). No IP was found in liver from oestrogen-treated rats or in ovaries from rats treated with luteinizing hormone. 3. 3) IP was found to have a molecular weight of 39,000 by sodium dodecyi sulphate-poly-acrylamide gel electrophoresis and an isoelectric point of 4.5 as determined by isoelectric focusing in polyacrylamide gels. No evidence for phosphorylation of IP was obtained. 4. 4) Extraction of uterine chromatin with 6 M urea, 0.4 M guanidinium chloride, 0.1 M sodium phosphate, pH 7.0, followed by sodium dodecyi sulphate-polyacrylamide gel electrophoresis indicated the presence of at least three oestradiol-induced proteins with molecular weights of 42,000, 25,000 and 16,000 dalton respectively. The component of molecular weight 42,000 which is closest in size to IP was present in only small amounts. None of these components were piesent in liver from oestrogen-treated rats. 5. 5) Maximal amounts of the oestrogen-induced chromosomal proteins and of IP were found when the uteri were explanted 1 h after intraperitoneal injection of oestradiol-17β. 6. 6) None of the oestradiol-induced proteins in uterine chromatin were histones, as judged from their molecular weight, but the component of molecular weight 16,000 was basic, as indicated by its adsorption to Bio-Rex-70 ion exchange resin.

Postnatal development of uterine response to estradiol-17β in the rat☆☆☆

Abstract The gradual acquisition of uterine responsiveness to a single injection of estradiol-17β was studied in rats aged 5–30 days with respect to wet weight and the content of DNA, RNA, and protein. No significant response in any of these parameters was found in 5- or 10-day-old rats. In 15- and 20-day-old rats all the parameters, except DNA content, were elevated by the hormone treatment; only in 30-day-old rats was there a significant increase in DNA content.

Synthesis of specific estradiol-induced protein by surviving rat uteri and cell-free uterine extracts☆

Abstract A cell-free system was developed for the synthesis of the specific ‘induced protein’ (IP) which is stimulated in the rat uterus within the first hour after treatment with estradiol-17β. Surviving uteri or uterine extracts were incubated with [3H]-labeled (estrogen-treated) or with [14C]- or [3S]-labeled (controls) leucine, glutamic acid or methionine, and the 150,000 gav supernatant solution was analyzed by electrophoresis on cellulose acetate. IP was detected by determining the isotope incorporation ratio. The 12,000 gmax post-mitochondrial supernatant suspension from uteri of 10- or 20-day old rats, prepared 1 h after injection of estradiol-17β (4 or 5 μg/rat), was found to synthesize IP. Treatment of surviving uteri with 30 nM estradiol-17β for l h at 37°C also permitted the extraction of a post-mitochondrial supernatant suspension capable of synthesizing IP. The electrophoretic distribution of radiolabeled soluble proteins was different in the cell-free system and surviving uteri. However, both preparations showed a single protein band with increased incorporation of tritiated amino acid after estradiol treatment. This band had an electrophoretic mobility relative to bovine serum albumin of approximately 1.1, characteristic of IP. Appearance of this band was prevented by addition of antibodies to estradiol or of cordycepin during incubation of the uteri with estradiol. The induction of IP in surviving uteri followed by IP synthesis in a cell-free system provides a versatile model for the more detailed analysis of the regulation of specific protein synthesis by estradiol.

Sequential Gene Expression in Response to Estradiol-l7β During Post-Natal Development of Rat Uterus

Hormonal model systems play an important role in the study of the control of protein synthesis (1) and of development (2). Thus the stimulation of nucleic acid and protein synthesis (3–5) in the immature rat uterus by treatment with estradiol–l7β has received considerable attention. Our recent use of a developmental approach (6–9) to the mechanism of action of estradio1–l7β provides a point of entry which complements and extends the current efforts through the study of receptor proteins and the control of RNA polymerase (10–12).

Specificities in the synthesis of a cytoplasmic estrogen-induced uterine protein.

The earliest known induction by estrogen of a specific uterine protein is that of the induced protein (IP) of Gorski and colleagues, which is demonstrable within 40 min after treatment. We found that this protein is not restricted to the rat; it was detected i the uterus of prepuberal C3H/eB and SWR mice, 1 h after injection of 4 B5g/rat) and coumestrol (300B5g/rat) and the synthetic estrogen diethylstilbestrol (5 B5/rat). We found no significant increase in the stimulation of the rate of IP synthesis by estrogen in uteri from either 10- or 20--day-old rats on subsequent treatment with actinomycin D, indicating that there was no superinduction of IP, despite a previous claim.