Analysis of basal regulatory elements in the 25-hydroxyvitamin D3 1alpha-hydroxylase gene promoter.

5'-Deletion analysis of the 1.7-kb mouse 1alpha-hydroxylase gene promoter reveals that the minimal promoter region for basal activity is -85/+22 and requires a functional CCAAT element. Mutational analysis also demonstrates that deletion of the internal promoter region from nucleotides -1125 to -86 leads to a 25- to 30-fold increase in basal promoter activity. The increased activity is not the result of positional effects, but is caused in part by the removal of an AC repeat. Further analysis of the promoter revealed an enhancer element localizes to an upstream region -1385 to -1125, which contains three consensus AP-1 sites. Deletion of the most proximal AP-1 site causes a 60% loss of enhancer activity. The data suggest the presence of the AC repeat prevents the full potential activation of the 1alpha-hydroxylase promoter by factors binding to AP-1 sites.

Regulation of 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression by parathyroid hormone and 1,25-dihydroxyvitamin D3.

The conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) takes place mainly in the kidney and is catalyzed by the enzyme 1alpha-hydroxylase. Parathyroid hormone (PTH) and 1,25-(OH)2D3 are well-known regulators of this tightly controlled step, but the mechanisms by which they modulate 1alpha-hydroxylase activity have not been fully delineated. Northern analysis showed PTH and forskolin rapidly and transiently increase 1alpha-hydroxylase expression in AOK-B50 cells and HKC-8 cells. Actinomycin D treatment blocks the increase, but cycloheximide does not. No decrease of 1alpha-hydroxylase transcript by 1,25-(OH)2D3 was observed in either cell line, although 24-hydroxylase levels were strongly induced by 1,25-(OH)2D3 treatment. 1,25-(OH)2D3 suppressed the 1alpha-hydroxylase transcript in vivo both in the presence and absence of exogenously supplied PTH. These results suggest that the stimulatory action of PTH is directly on the 1alpha-hydroxylase gene, while the repressive action of 1,25-(OH)2D3 does not involve the parathyroid gland but is nevertheless indirect.

Parathyroid hormone activation of the 25-hydroxyvitamin D3-1alpha-hydroxylase gene promoter.

The DNA flanking the 5' sequence of the mouse 1alpha-hydroxylase gene has been cloned and sequenced. A TATA box has been located at -30 bp and aCCAAT box has been located at -79 bp. The gene's promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D3 does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D3-1alpha-hydroxylase by acting on the promoter of the 1alpha-hydroxylase gene.

Increased latent gelatinase activity in the sclera of visually deprived chicks.

PURPOSE: Gelatinase activity was measured in the normal chick sclera and in sclera of form-deprived (myopic) eyes to assess the role of this metalloproteinase in ocular elongation associated with experimental myopia. METHODS: Gelatinases were extracted from anterior and posterior regions of normal chick sclera and sclera from eyes that had been form-vision deprived for 11 days. Gelatinase activity in the extracts was determined by measuring the digestion of 3H-gelatin after incubation with the extracts in the absence or presence of 1 mM aminophenylmercuric acetate (APMA) to activate latent gelatinases. Scleral gelatinases were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gelatin zymography and immunoprecipitation analyses. RESULTS: No significant differences were detected in gelatinase activity between normal and deprived posterior sclera in the absence of APMA. However, when scleral extracts were incubated with APMA, extracts from the posterior sclera of deprived eyes contained significantly more gelatinase activity than paired controls (+127%, P = 0.0105). In contrast, no differences in active or latent gelatinase activity were detected in extracts from the anterior sclera. Removal of tissue inhibitors of metalloproteinases (TIMP) from control scleral extracts by reduction and alkylation resulted in a 222% increase in gelatinolytic activity after APMA-activation (P < or = 0.001), whereas similar treatment of deprived scleral extracts resulted in only a 76% increase in gelatinolytic activity (P < or = 0.001). A 65/58-kd doublet was the major gelatinolytic species from control and deprived posterior sclera that represent the proenzyme and active forms of the 72-kd gelatinase (MMP-2). CONCLUSIONS: These data indicate that visual deprivation is associated with an increased amount of the 72-kd progelatinase and a decreased amount of TIMP within the posterior sclera. Therefore, an imbalance between the levels of 72-kd progelatinase and its inhibitor may play a role in the remodeling processes of the posterior sclera during the development of form-deprivation myopia.