M. Elizabeth Bruns

Age and gender effects on 1,25-dihydroxyvitamin D3-regulated gene expression.

Several factors involved in regulation of bone mineral metabolism were compared in male and female Fischer 344 rats of different ages (1, 2.5, 6, and 18 months). Plasma 1,25-(OH)2D3 concentrations decreased with age in rats of both genders. Abundance of calbindin-D28K and its mRNA in kidney and calbindin-D9K and its mRNA in duodenum also decreased with age in both male and female rats. Renal 24-hydroxylase activity and 24-hydroxylase mRNA content were elevated significantly in 18-month-old males and females, compared with younger ages. These data suggest that increased renal catabolism of 1,25-(OH)2D3 may be responsible for low plasma 1,25-(OH)2D3 concentrations observed in older animals. Plasma PTH and 1,25-(OH)2D3 concentrations, renal 24-hydroxylase enzyme activity and 24-hydroxylase mRNA content, duodenal 24-hydroxylase mRNA abundance, and duodenal calbindin-D9K and calbindin-D9K mRNA content were greater in males than in females at 2.5 months of age. Lower plasma 1,25-(OH)2D3 concentrations in females seem to explain observed gender differences in expression of 1,25-(OH)2D3-stimulated genes. The combined effects of these gender differences at ages when peak bone density is being developed may contribute to the greater incidence of osteoporosis in females than in males.

Expression of parathyroid hormone – related protein and its receptor in human umbilical cord: Evidence for a paracrine system involving umbilical vessels

OBJECTIVE Our purpose was to study the expression and localization of parathyroid hormone-related protein and expression of the parathyroid hormone/parathyroid hormone-related protein receptor in human umbilical cord. STUDY DESIGN The expression and localization of parathyroid hormone-related protein and expression of the parathyroid hormone/parathyroid hormone-related protein receptor were studied in isolated tissues from the human umbilical cord by Northern analysis, reverse-transcriptase polymerase chain reaction, Southern gel analysis, and immunolocalization procedures at the light and electron microscopic levels. RESULTS Parathyroid hormone-related protein was abundantly expressed in the umbilical cord. Immunohistochemical and immunocytochemical techniques confirmed hormone localization in the amnion epithelial layer and in vascular smooth muscle and endothelial cells in vessels from the umbilical cord and placental chorionic plate. Multiplex reverse-transcriptase polymerase chain reaction identified expression of receptor messenger ribonucleic acid in vessels of the umbilical cord; this finding was verified by means of Southern gel analysis of the products of the reverse-transcriptase polymerase chain reaction. CONCLUSION A parathyroid hormone-related protein paracrine system appears to exist in human umbilical cord. We suggest that it may be involved in the control of fetal placental circulation.

Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acid in human amnion and chorion-decidua: Implications for secretion and function

OBJECTIVE Our purpose was to define the location and packaging of parathyroid hormone-related peptide in amnion-chorion and the potential target tissues for its action in fetal membranes. STUDY DESIGN We studied fetal membranes by use of light microscopic immunocytochemistry with three monoclonal antibodies against distinct regions of the parathyroid hormone-related peptide molecule. For electron microscopy immunogold analysis with a monoclonal antibody specific to the 109-141 fragment was used to observe parathyroid hormone-related peptide intracellularly in amnion membrane and in the chorion layers. Multiplex reverse transcriptase-polymerase chain reaction with Southern blotting was used to identify parathyroid hormone/parathyroid hormone-related peptide receptor and control messenger ribonucleic acids in amnion and chorion-decidua. RESULTS All monoclonal antibodies revealed immunoreactive parathyroid hormone-related peptide in the amniotic epithelial cells and in some fibroblast-like cells embedded in the extracellular matrix of the amnion. Parathyroid hormone-related peptide was also found in the chorion in fibroblast and trophoblast layers and in decidua. Ultrastructurally immunogold particles were evenly distributed throughout the amniotic epithelial cells and were present in apical microvilli and near the basal membranes. Electron microscopy studies of the chorion cytotrophoblast also showed freely dispersed immunogold particles of parathyroid hormone-related peptide with no packaging in secretory granules. Low to undetectable levels of parathyroid hormone/parathyroid hormone-related peptide receptor messenger ribonucleic acid were found in amnion tissue, whereas abundant receptor messenger ribonucleic acid was found in chorion-decidua. CONCLUSIONS These results suggest the presence of a parathyroid hormone-related peptide paracrine system within the human fetal membranes.

The parathyroid hormone-related protein (PTHrP) gene preferentially utilizes a GC-rich promoter and the PTHrP 1-139 coding pathway in normal human amnion

Parathyroid hormone-related protein (PTHrP) is an oncofetal protein that is widely expressed in mammalian tissues. The PTHrP gene is a complex with three transcriptional start-sites, two TATA boxes and a GC-rich region, and three predicted polypeptide products, PTHrP 1-141, PTHrP 1-139, and PTHrP 1-173. The originally discovered form of PTHrP, PTHrP 1-141, and the classical TATA box promoters are generally assumed to be the major pathways of PTHrP gene expression. We have used the polymerase chain reaction (PCR) to study PTHrP gene expression in the human amnion. Our studies demonstrate that the GC-rich promoter is preferentially used and that PTHrP 1-139 is the major PTHrP mRNA expressed in human amnion. PTHrP 1-139 lacks the carboxy-terminal arginine and histidine residues of PTHrP 1-141; these two basic amino acids could have significant effects on the biological activity of PTHrP. These preferential pathways for PTHrP gene expression are shared by malignant and normal human tissues.

Expression and specific immunolocalization of the human parathyroid hormone/parathyroid hormone–related protein receptor in the uteroplacental unit

OBJECTIVE Our purpose in these studies was to determine the expression and cellular localization of the parathyroid hormone/parathyroid hormone-related protein receptor in the human uteroplacental unit. STUDY DESIGN Human uteroplacental tissues were obtained and ribonucleic acid was extracted. Reverse transcriptase-polymerase chain reaction was performed with use of primers for both the parathyroid hormone/parathyroid hormone-related protein receptor and human phosphoglyceraldehyde dehydrogenase. Ethidium bromide-stained gels and Southern blots were evaluated, and polymerase chain reaction fragments were sequenced. For immunohistochemistry, slides were incubated with a newly developed antibody (3D1.1) specific for the parathyroid hormone/parathyroid hormone-related protein receptor, and bound monoclonal antibody was detected by use of the avidin-biotin technique. RESULTS Reverse transcriptase polymerase chain reaction gels and blots showed that receptor messenger ribonucleic acid was present in choriodecidua, placenta, and myometrium. Sequence analysis revealed complete identity of the receptor product and the known nucleotide sequence in the receptor. There was intense receptor staining of the myometrial smooth muscle as well as staining of the endothelium and smooth muscle of the associated vasculature. In umbilical cord immunoreactive receptor was found in the vascular endothelium and vascular smooth muscle cells and in stromal cells. In choriodecidua receptor was found in chorionic trophoblasts and decidualized endometrial stromal cells. In all tissues immunostaining was specific, as evidenced by the blocking of staining after addition of receptor peptide to the antibody (absorbed controls). CONCLUSION The parathyroid hormone/parathyroid hormone-related protein receptor is widely expressed in the human uteroplacental unit. The cellular localizations of the receptor in smooth muscle reflect the ability of parathyroid hormone-related protein to relax both uterine and vascular smooth muscle. The presence of novel autocrine and paracrine systems in the human uteroplacental unit is suggested by the finding that the same cells or adjacent cells produce both parathyroid hormone-related protein and its receptor.

Expression of Parathyroid Hormone-Related Peptide (PTH-rp) and Its Receptorin the Porcine Ovary: Regulation by Transforming Growth Factor-β and Possible Paracrine Effects of Granulosa Cell PTH-rp Secretion on Theca Cells

Abstract Parathyroid hormone-related peptide (PTH-rp) and the PTH-rp receptor are expressed in certain cancers as well as in many normal tissues. To evaluate the expression of this Ca2+-regulating hormone and its receptor in porcine ovary, we isolated partial cDNAs encoding homologous PTH-rp and PTH-rp receptor using reverse transcription-polymerase chain reaction (RT-PCR). The cDNA encoding PTH-rp (419 base pairs [bp]) was 92% and 87% homologous to human and rat sequences, respectively, while the PTH-rp receptor clone (167 bp) was 94% and 91% identical to the human and rat genes. Qualitative estimates of PTH-rp mRNA by RT-PCR indicated that the PTH-rp gene is expressed at high levels in the corpus luteum but is undetectable in granulosa and theca cells isolated from small (1–5 mm) and medium-sized (5–8 mm) antral follicles. In contrast, PTH-rp receptor transcripts were most abundant in corpora lutea and theca cells, and least abundant (albeit detectable) in granulosa cells. Regulation of PTH-rp protein production was assessed in serum-free monolayer cultures of porcine granulosa cells. Transforming growth factor (TGF)-β1 (100 ng/ml) increased PTH-rp concentrations (assayed by two-site immunoradiometric assay of culture media) as well as corresponding PTH-rp mRNA accumulation (assessed by RT-PCR) in a time-dependent manner, with maximal responses of 3- to 5-fold at 96 h. TGF-β1 dose-response studies revealed an ED50 of 0.24–0.38 ng/ml with a maximal effect at 30 ng/ml. Other growth factors and hormones, including insulin, insulin-like growth factor (type I), epidermal growth factor, FSH, estradiol, and interleukin-1, failed to alter PTH-rp secretion. Biological effects of PTH-rp were evident in purified porcine theca cells. Using the Ca2+-sensitive fluorescent indicator dye, fura-2, and digital imaging videomicroscopy, we found that PTH-rp (1 μM) stimulated intracellular free calcium ion concentrations ([Ca2+]i) in single porcine theca cells. The [Ca2+]i elevation was characterized by a slow and prolonged rise. After PTH-rp stimulation, theca cells maintained responsiveness to hormone stimulation by LH, which elicited a typical theca cell [Ca2+]i response. Our results allow a hypothesis of a paracrine intrafollicular signaling system involving interaction between theca cell-derived TGF-β and granulosa cell-derived PTH-rp, with feedback by PTH-rp on theca cells. Alternatively, expression of mRNAs encoding PTH-rp and its receptor in corpora lutea suggests that this peptide may play a role in luteal cell function. The precise role of this intraovarian PTH-rp system will require further study.

Interleukin-1β and interleukin-4 increase parathyroid hormone—related protein secretion by human umbilical vein endothelial cells in culture

Abstract OBJECTIVE: Our purpose was to learn whether cytokines such as interleukin-1β and related (or antagonistic) cytokines, hormones, and growth factors could regulate secretion of the vasorelaxant parathyroid hormone—related protein in human umbilical vein endothelial cells in culture. STUDY DESIGN: Secondary cultures of human umbilical vein endothelial cells were grown to confluence and treated with interleukin-1β, an array of factors with possible regulatory actions (cytokines, growth factors, vasoactive peptides, and steroids), and a phorbol ester as a stimulatory control. After 24 hours immunoreactive parathyroid hormone—related peptide in the media was measured by a two-site sandwich radioimmunoassay. The mechanism of interleukin-1β action was probed with interleukin-1β receptor antagonist and selected inhibitors. RESULTS: Interleukin-1β (10 ng/ml) produced up to an eightfold increase in parathyroid hormone—related peptide secretion from human umbilical vein endothelial cells in culture ( p p −7 mol/L significantly increased the secretion of parathyroid hormone—related peptide by human umbilical vein endothelial cells ( p CONCLUSION: Interleukin-1β and interleukin-4 increase parathyroid hormone—related protein secretion in human umbilical vein endothelial cells in culture. Because interleukin-1β messenger ribonucleic acid has been found in umbilical cord endothelial cells, we propose that the umbilical cord has a novel vasorelaxant regulatory system that uses interleukin-1β endothelial action and secretion of the vasorelaxant parathyroid hormone—related peptide.

Calbindin-D9k Localization and Gestational Changes in the Utero-Placental Unit: A Model for Maternal-Fetal Calcium Transports

The calbindins are a class of E-F hand, high affinity calcium-binding proteins. In a variety of tissues, the synthesis of these proteins is known to be dependent on 1,25dihydroxyvitamin D (1,25 (OH)2D) (Christakos et al., Wasserman et al). Two major forms of calbindins have been isolated and characterized, namely a high molecular weight form of Mr 28,000 (calbindin-D28k) first isolated from chick intestinal epithelium, and a low molecular weight form of Mr 9,000 (calbindin-D9k) first identified in rat intestinal mucosa. These proteins show distinct differences in their distributions, both among species and among tissues. Although the molecular role of these proteins is as yet unclear, ample evidence indicates that calbindins are an excellent marker for epithelia that sustain high rates of transcellular calcium transport (Christakos et al., Wasserman et al.)

ATP-DEPENDENT CALCIUM UPTAKE BY DUODENAL BASOLATERAL MEMBRANES FROM DEVELOPING RATS11Supported by NIH Grants HD-12335 (MEB) and AM-25897 (JMM).

Publisher Summary This chapter discusses the appearance of active calcium absorption at the time of weaning that might reflect a vitamin D-regulated induction of the ATP-dependent calcium pump. In rats prior to weaning, calcium absorption is entirely passive, whereas an active calcium absorption appears at weaning. Recently, vitamin D has been reported to increase the activity of an active, ATP-dependent calcium pump in the basolateral plasma membrane of rat intestine. Basolateral plasma membranes were isolated from washed enterocytes by differential and density gradient centrifugations. Basolateral membranes from preweaned (2-week old) and weaned (5-week old) animals were comparably enriched in marker enzymes including Na, K-ATPase and disaccharide. Calcium uptake by basolateral membranes was unaffected by weaning. The apparent effect of calcitriol on the calcium pump may have reflected a response to increased extracellular calcium. The weanling rat provides a model for study of the intestinal effects of vitamin D. At weaning, despite increases in plasma calcitriol, serum calcium changes little.