Regulation of cutaneous previtamin D3 photosynthesis in man: skin pigment is not an essential regulator.

When human skin was exposed to simulated solar ultraviolet radiation, epidermal 7-dehydrocholesterol was converted to previtamin D3. During prolonged exposure to simulated solar ultraviolet radiation, the synthesis of previtamin D3 reached a plateau at about 10 to 15 percent of the original 7-dehydrocholesterol content, and previtamin D3 was photoisomerized to two biologically inert isomers, lumisterol3 and tachysterol3. Increases either in skin melanin concentration or in latitude necessitated increases in the exposure time to simulated solar ultraviolet radiation required to maximize the formation, but not the total content, of previtamin D3. In order of importance, the significant determinants limiting the cutaneous production of previtamin D3 are (i) photochemical regulation, (ii) pigmentation, and (iii) latitude.

Spectral character of sunlight modulates photosynthesis of previtamin D3 and its photoisomers in human skin.

The photosynthesis of previtamin D3 from 7-dehydrocholesterol in human skin was determined after exposure to narrow-band radiation or simulated solar radiation. The optimum wavelengths for the production of previtamin D3 were determined to be between 295 and 300 nanometers. When human skin was exposed to 295-nanometer radiation, up to 65 percent of the original 7-dehydrocholesterol content was converted to previtamin D3. In comparison, when adjacent skin was exposed to simulated solar radiation, the maximum formation of previtamin D3 was about 20 percent. Major differences in the formation of lumisterol3, and tachysterol3 from previtamin D3 were also observed. It is concluded that the spectral character of natural sunlight has a profound effect on the photochemistry of 7-dehydrocholesterol in human skin.

Photosynthesis of previtamin D3 in human skin and the physiologic consequences.

Photosynthesis of previtamin D3 can occur throughout the epidermis in the dermis when hypopigmented Caucasian skin is exposed to solar ultraviolet radiation. Once previtamin D3 is formed in the skin, it undergoes a temperature-dependent thermal isomerization that takes at least 3 days to complete. The vitamin D-binding protein preferentially translocates the thermal product, vitamin D3, into the circulation. These processes suggest a unique mechanism for the synthesis, storage, and slow, steady release of vitamin D3 from the skin into the circulation.

Aging decreases the capacity of human skin to produce vitamin D3.

An evaluation of surgically obtained skin (age range, 8-92 yr) revealed that there is an age-dependent decrease in the epidermal concentrations of provitamin D3 (7-dehydrocholesterol). To ascertain that aging indeed decreased the capacity of human skin to produce vitamin D3, some of the skin samples were exposed to ultraviolet radiation and the content of previtamin D3 was determined in the epidermis and dermis. The epidermis in the young and older subjects was the major site for the formation of previtamin D3, accounting for greater than 80% of the total previtamin D3 that was produced in the skin. A comparison of the amount of previtamin D3 produced in the skin from the 8- and 18-yr-old subjects with the amount produced in the skin from the 77- and 82-yr-old subjects revealed that aging can decrease by greater than twofold the capacity of the skin to produce previtamin D3. Recognition of this difference may be extremely important for the elderly, who infrequently expose a small area of skin to sunlight and who depend on this exposure for their vitamin D nutritional needs.

Sunscreens suppress cutaneous vitamin D3 synthesis.

Sunscreens block the cutaneous absorption of UV-B radiation and prevent sunburning, premature aging, and cancer of the skin. Inasmuch as UV-B radiation is also responsible for the photosynthesis of vitamin D3, we investigated the effect of sunscreens on the cutaneous formation of vitamin D3 in vivo and in vitro. Eight normal subjects, four of whom had been protected with the sunscreen para-aminobenzoic acid (sun protection factor 8), were exposed to one minimal erythema dose of UV radiation. The mean serum vitamin D3 concentration increased from 1.5 +/- 1.0 (+/- SEM) to 25.6 +/- 6.7 ng/mL in unprotected subjects, whereas it was 5.6 +/- 3.0 and 4.4 +/- 2.4 ng/mL at these times in the subjects who were protected with para-aminobenzoic acid. Para-aminobenzoic acid also prevented the photoisomerization of 7-dehydrocholesterol to previtamin D3 in human skin slices in vitro. These results indicate that the sunscreen interferred with the cutaneous production of vitamin D3.

Cultured human keratinocytes cannot metabolize vitamin D3 to 25-hydroxyvitamin D3.

The metabolism of [3H]vitamin D3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [3H]vitamin D3 and the lipid soluble fractions from the media and cells were extracted by high-performance liquid chromatography (HPLC). Vitamin D3 and its metabolites, 25-OH-D3, 24,25(OH)2D3 and 1,25(OH)2D3 were added to the extracts, as markers, prior to HPLC. HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [3H]25-OH-D3 showed a 10 +/- 4% conversion to [3H]1,25(OH)2D3 whereas no conversion to [3H]1,25(OH)2D3 was observed in control CHKs that were boiled prior to incubation with [3H]25-OH-D3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D3 to 25-OH-D3.

1,25(OH)2D3 increases calcium and phosphatidylinositol metabolism in differentiating cultured human keratinocytes.

The effect of 1,25(OH)(2)D(3) on the intracellular calcium, (Ca(+2))i, in both cultured human keratinocytes and in cultured human dermal fibroblasts was investigated. When the intracellular calcium (Ca(+2))i in cultured human keratinocytes, grown in a serum-free medium containing 1.8 mM calcium, was measured by the fluorescent calcium-indicator, Furu-2, the (Ca(+2)i increased 154%, 202%, and 409% over the control value after incubation with 1,25(OH)(2)D(3) at 10(-10) m, 10(-8) m, and 10(-6) m, respectively. This response was immediate (15 seconds), specific (no effect with either 25(OH)D(3) at 10(-8) m or vitamin D(3) at 10(-8) m), and occurred with or without EGTA in the medium. In contrast, 1,25(OH)(2)D(3) did not increase the (Ca(2+))i in either cultured human keratinocytes that were grown in low calcium (0.05 mm), serum-free medium or in cultured human dermal fibroblasts that were grown in medium containing 0.05 mm calcium and 1% serum. The effect of 1,25(OH)(2)D(3) on the the turnover of phosphatidylinositol was investigated as a possible cause for the observed increase in (Ca(+2)i. Cultured human keratinocytes that were incubated with (3)H-inositol demonstrated a 50 % +/- 10% increase in the triphosphated, plasma membrane-bound metabolite of phosphatidylinositol, PIP(2), by 15 seconds, followed by a rapid decrease at 30 seconds, then a return toward basal levels by 1 minute. Lysophosphatidylinositol, which results from the sn-2 deacylation of phosphatidylinositol by phospholipase A(2), decreased 20% +/- 8% within 30 seconds, then increased to 200% +/- 10% of the control value by 5 minutes. The accumulation of IP(3) was increased 50% to 100% above the control value within 30 seconds and this increase was substained during the 5-minute incubation period. Stimulation of phosphatidylinositol turnover by 1,25(OH)(2)D(3) was not detected in either cultured human keratinocytes that were grown in serum-free, low calcium medium or in cultured human dermal fibroblasts that were grown in 1% serum.

Ribonucleic acid synthesis by spermatozoa from the rat and hamster.

Spermatozoa from the cauda of the epididymis of the hamster and rat were incubated with [5-(3)H]uridine and glucose. By using a procedure avoiding bacterial and other cellular contamination, sonic extracts were prepared and digested with deoxyribonuclease and Pronase. Radioactive RNA of high molecular weight was isolated by two methods: (a) gel filtration on Sephadex G-75 columns and (b) polyacrylamide-gel electrophoresis in which it migrated in the region of 28S and 23S RNA markers. The macromolecules were alkali-labile and hydrolysed by ribonuclease. From (3)H radioactivity and E(260) of the isolated RNA the rate of incorporation of uridine into RNA of spermatozoa was calculated to be 0.1-0.5nmol/h per mg of RNA.

Changes in lipase and phosphatase activities of rat spermatozoa in transit from the caput to the cauda epididymidis.

Rat spermatozoa from the cauda epididymidis, freed from their cytoplasmic droplets and acrosomes, were found to have a lower lipid content and to incorporate [14C]glucose into their glycerides and glycerophosphatides at a lower rate than spermatozoa from the caput epididymidis. Against the background of the activities of some glycolytic enzymes which remained constant the activity of alkaline phosphatase decreased in spermatozoa migrating through the epididymis, whereas the activity of monoglyceride lipase increased. The corresponding enzyme activities of non-flagellate germ cells of the testis were measured for comparison. The triglyceride lipase of non-flagellate germ cells and of spermatozoa from both caput and cauda epididymidis was activated by cyclic 3':5'-AMP.

24-Dehydrovitamin D is potent inhibitor of rat liver microsomal vitamin D-25-hydroxylase.

A pathway has been described in the skin for the synthesis of 24-dehydrovitamin D3 (delta 24D3) from 24-dehydroprovitamin D3. The physiologic function of delta 24D3 is unknown, but has been proposed as a potential inhibitor of hepatic vitamin D-25-hydroxylase. We validated an assay for vitamin D-25-hydroxylase in rat hepatic microsomes, using nanomolar amounts of [3H]D3 as substrate, and found that delta 24D3 competitively inhibits vitamin D-25-hydroxylase activity. The apparent Ki was approximately 17 nM, indistinguishable from the Km of approximately 15 nM, suggesting that both delta 24D3 and cholecalciferol have similar affinity for the enzyme. We found no [3H]delta 24D3 in serum or liver extracts after repletion of vitamin D-depleted rats with [3H]vitamin D3 for 4 h or 6 days. A dose of 1 microgram delta 24D3 to vitamin D- and calcium-depleted rats was unable to promote any elevation in the 45Ca transport by everted duodenal sacs or to increase levels of plasma calcium: thus no evidence for biological conversion of delta 24D3 to vitamin D3 was observed. Further studies are needed to determine whether delta 24D3 is released from the skin to the circulation and is taken up by the liver, before physiological relevance can be attributed to this inhibitor.

Cultured psoriatic fibroblasts from involved and uninvolved sites have a partial but not absolute resistance to the proliferation-inhibition activity of 1,25-dihydroxyvitamin D3.

We examined the responsiveness of cultured dermal fibroblasts from biopsies of uninvolved and involved areas of skin from six patients with psoriasis to the cell-proliferation-inhibition activity of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3). Cultured fibroblasts from age-matched controls responded to 1,25-(OH)2-D3 (at 0.01, 1, 10, and 100 microM) in a dose-dependent fashion, whereas cultured psoriatic fibroblasts from involved or uninvolved skin showed no inhibition of proliferation when exposed to 0.01 or 1 microM of 1,25-(OH)2-D3. However, 1,25-(OH)2-D3 did inhibit proliferation of cultured psoriatic fibroblasts when the concentrations were increased to 10 and 100 microM. An analysis of the 1,25-(OH)2-D3 receptors in cultured psoriatic fibroblasts from uninvolved skin revealed that the Kd, nmax, and sedimentation coefficient were identical to the receptors found in the fibroblasts from age-matched controls. Therefore, cultured psoriatic fibroblasts from involved and uninvolved skin have a partial resistance to 1,25-(OH)2-D3, suggesting that there may be a biochemical defect that is inherent in the dermal fibroblasts of psoriatic patients. Recognition of this defect may provide a new approach for the evaluation of the cause and treatment of this disfiguring skin disorder.

The photoproduction of 1 alpha,25-dihydroxyvitamin D3 in skin: an approach to the therapy of vitamin-D-resistant syndromes.

Cutaneous 7-dehydrocholesterol, exposed to ultraviolet radiation, converts to previtamin D3, which in turn converts in skin to vitamin D3 and is carried into the circulation. We investigated the feasibility of the photochemical conversion in skin of hydroxylated derivatives of 7-dehydrocholesterol - such as 1 alpha, 25-dihydroxy-7-dehydrocholesterol (1 alpha, 25-(OH)2-7-DHC) - to the corresponding hydroxylated previtamin as an alternative method of delivery of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2-D3) to subjects who are deficient in the endogenous metabolite. In human volunteers and in vitamin-D-deficient rats [24-3H] 1 alpha, 25-(OH)2-D3 appeared in blood after [24-3H] 1 alpha, 25-(OH)2-7-DHC was applied to the skin and exposed to ultraviolet radiation. In anephric rats, intestinal calcium absorption and serum calcium levels were elevated after a topical dose of 1 alpha, 25-(OH)2-7-DHC combined with ultraviolet phototherapy. Delivery of equivalent doses of 1 alpha, 25-(OH)2-D3 through the skin and orally showed that there was more prolonged stimulation in intestinal calcium absorption and serum calcium elevation after cutaneous administration. The photochemical conversion of precursors may be useful in the treatment of patients with impaired vitamin-D metabolism.

The paternal allele of the H19 gene is progressively silenced during early mouse development: the acetylation status of histones may be involved in the generation of variegated expression patterns.

Transcriptional silencing can reflect heritable, epigenetic inactivation of genes, either singly or in groups, during the life-time of an organism. This phenomenon is exemplified by parent-of-origin-specific inactivation events (genomic imprinting) for a subset of mammalian autosomal genes, such as H19. Very little is known, however, about the timing and mechanism(s) of silencing of the paternal H19 allele during mouse development. Using a novel in situ approach, we present evidence that the silencing of the paternal H19 allele is progressive in the trophectodermal lineage during early mouse development and generates variegated expression patterns. The silencing process apparently involves recruitment of histone deacetylases since the mosaic paternal-specific H19 expression reappears in trichostatin A-treated mouse conceptuses, undergoing in vitro organogenesis. Moreover, the paternal H19 alleles of PatDup.d7 placentas, in which a region encompassing the H19 locus of chromosome 7 is bipaternally derived, partially escape the silencing process and are expressed in a variegated manner. We suggest that allele-specific silencing of H19 share some common features with chromatin-mediated silencing in position-effect variegation.