Parathyroid hormone (PTH) exerts both catabolic and anabolic actions on bone. Studies on the skeletal effects of PTH have seldom considered the effects of gender. Our study was designed to determine whether the response of mouse bone to PTH differed according to sex. As a first step, we analyzed gender differences with respect to bone mass and structural properties of 4 month old PTH treated (80 µg/kg per day for 2 weeks) male and female CD-1 mice. PTH significantly increased fat free weight/body weight, periosteal bone formation rate, mineral apposition rate, and endosteal single labeling surface, while significantly decreasing medullary area in male mice compared with vehicle treated controls, but induced no significant changes in female mice. We then analyzed the gender differences in bone marrow stromal cells (BMSC) isolated from 4 month old male and female CD-1 mice following treatment with PTH (80 µg/kg per day for 2 weeks). PTH significantly increased the osteogenic colony number and the alkaline phosphatase (ALP) activity (ALP/cell) by day 14 in cultures of BMSCs from male and female mice. PTH also increased the mRNA level of receptor activator of nuclear factor B ligand in the bone tissue (marrow removed) of both females and males. However, PTH increased the mRNA levels of IGF-I and IGF-IR only in the bones of male mice. Our results indicate that on balance a 2-weeks course of PTH is anabolic on cortical bone in this mouse strain. These effects are more evident in the male mouse. These differences between male and female mice may reflect the greater response to PTH of IGF-I and IGF-IR gene expression in males enhancing the anabolic effect on cortical bone.
Vitamin D 3 is synthesized in the skin, undergoes 25-hydroxylation catalyzed by vitamin D 3 -25-hydroxylase in the liver (1, 2), followed by 1␣-hydroxylation in the kidney and other tissues to make the biologically active hormone 1,25(OH) 2 D 3 . The 1␣-hydroxylase 1 activity is catalyzed by mitochondrial P450c1␣, encoded by a gene termed CYP27B1 (3)(4)(5). This is an activation pathway for vitamin D metabolites. 25(OH)D 3 and 1,25(OH) 2 D 3 can then be converted to the less active forms 24,25(OH) 2 D 3 and 1,24,25(OH) 3 D 3 , respectively, by the 24-hydroxylase activity of P450c24 encoded by a gene termed CYP24 (6, 7). These are inactivation pathways for vitamin D metabolites. The most important site of 1␣-hydroxylase and 24-hydroxylase activities is in proximal renal tubular cells, but these enzymes are also found in various extrarenal tissues including epidermal keratinocytes (4,8,9 may not regulate the 1␣-hydroxylase by such a mechanism. To test this hypothesis, we examined the effect of exogenous 1,25(OH) 2 D 3 on the abundance of 1␣-hydroxylase mRNA and protein in cultured normal human keratinocytes compared with the apparent activity of the 1␣-hydroxylase under conditions in which 24-hydroxylase was inhibited. We found that 1,25(OH) 2 D 3 did not inhibit 1␣-hydroxylase enzyme activity or the abundance of its mRNA or protein. Rather, the feedback regulation of 1,25(OH) 2 D 3 involves its catabolism by 24-hydroxylase induction instead of its synthesis by 1␣-hydroxylase. EXPERIMENTAL PROCEDURESCell Culture-Normal human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM, Clonetics, San Diego, CA) as previously described (19). Briefly, keratinocytes were isolated from newborn human foreskins by trypsinization (0.25% trypsin, 4°C, overnight), and primary cultures were established in KGM containing 0.07 mM calcium. First and second passage keratinocytes were plated with KGM containing 0.03 mM calcium and used in the subsequent experiments described.Enzyme Activity Assays-To measure 1␣-hydroxylase and 24-hydroxylase activities, we used the method of Bikle et al. (8) (Amersham Biosciences) was added to cultured normal human keratinocytes in 6-well plates. Following 1 h of incubation at 37°C, the reaction was stopped with 1 ml of methanol. Both cells and medium were extracted by the method of Bligh and Dyer (20). Metabolites in the chloroform extract were separated and quantitated by a Waters high performance liquid chromatography (HPLC) system (Waters Associates, Milford, MA) linked to a Flow Scintillation Analyzer (Packard, Meriden, CT). HPLC utilized a DuPont Zorbax Sil column (4.6 ϫ 25 cm) and a non-linear concave gradient from 97:3 to 90:10 hexane:isopropanol for 1␣-hydroxylated steroids or 90:10 hexane:isopropanol for 24-hydroxylated steroids. Output was monitored by radioactivity with a Flow Scintillation Analyzer (Packard, Downers Grove, IL). Chemically synthesized standards were used to determine the elution volumes of the metabolites.RNA Anal...
We recently identified a 28-kDa protein in the intestinal brush border that resembled tropomyosin in terms of size, homology, and ␣ helical content. This protein contained 27 heptad repeats, nearly all of which began with leucine, leading to its name zipper protein. Subsequent analysis, however, indicated that both a 49-kDa and a 28-kDa immunoreactive protein existed in intestinal brush-border extracts. Using 5-rapid amplification of cDNA ends analysis, we extended the N-terminal sequence of zipper protein to the apparent translation start site. This additional sequence contained a putative transmembrane domain and two potential tryptic cleavage sites C-terminal to the transmembrane domain which would release a 28-kDa cytoplasmic protein if utilized. The additional sequence was highly homologous to members of the B-G protein family, a family with no known function. Immunoelectron microscopy showed that zipper protein was confined to the membrane of the microvillus where it was in close association with brush-border myosin 1 (BBM1). Recombinant zipper protein (28-kDa cytoplasmic portion) blocked the binding of actin to BBM1 and inhibited actin-stimulated BBM1 ATPase activity. In contrast, zipper protein had no effect on endogenous or K/EDTA-stimulated BBM1 ATPase activity. Furthermore, zipper protein displaced tropomyosin from binding to actin, suggesting that these homologous proteins bind to the same sites on the actin molecule. We conclude that zipper protein is a transmembrane protein of the B-G family localized to the intestinal epithelial cell microvillus. The extended cytoplasmic tail either in the intact molecule or after tryptic cleavage may participate in regulating the binding and, thus, activation of BBM1 by actin in a manner similar to tropomyosin.
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