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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have evaluated the effects of retinoic acid as a differentiating agent on two pluripotential mesenchymal stem cell lines, the mouse cell line C3H-10T1/2 (10T1/2), which has the capacity to differentiate in vitro into myoblasts, adipocytes, chondrocytes, and osteoblasts, and the rat cell line ROB-C26 (C26), which can, in culture, give rise to adipocytes, myoblasts, and osteoblasts. Retinoic acid (10(-6) M) reduces the incidence of myoblast and adipocyte formation and induces or increases alkaline phosphatase expression and responsiveness to PTH, two indicators of the osteoblastic phenotype. Because transforming growth factor-beta (TGF beta) superfamily members, including the different TGF beta isoforms and the bone morphogenetic proteins (BMPs), are thought to play a role in regulating bone and cartilage formation, and because exogenous TGF beta and BMP-2 have already been found to modulate osteoblastic differentiation of C26 and 10T1/2 cells, we evaluated the endogenous expression of these factors in both cell lines cultured in the presence or absence of retinoic acid. Our data show that C26 and 10T1/2 cells constitutively express a broad spectrum of TGF beta superfamily members. However, this pattern of expression is dramatically altered in response to retinoic acid. Specifically, expression of TGF beta 1 and especially TGF beta 2 is strongly increased, whereas TGF beta 3 expression is down-regulated. These changes are accompanied by a striking decline in TGF beta receptor expression levels at the cell surface. Furthermore, BMP-2 and -4 expression are decreased after treatment with retinoic acid, whereas vgr-1/BMP-6 expression is induced in C26 cells, but decreased in 10T1/2 cells. These results clearly show a dynamic changing pattern of TGF beta superfamily expression consequent to the induction of osteogenic differentiation and provide the first indication that TGF beta receptor down-regulation may be an essential part of this differentiation process. These data also establish the C26 and 10T1/2 cell lines as convenient in vitro model systems for exploring the autoregulation of osteogenic differentiation by members of the TGF beta superfamily.
Mol Endocrinol 1993 Feb
PMID:Modulation of expression and cell surface binding of members of the transforming growth factor-beta superfamily during retinoic acid-induced osteoblastic differentiation of multipotential mesenchymal cells. 838 38

Incubation of bovine parathyroid cells for 48 h in 0.4 mmol calcium/l had no significant effect on steady-state preproparathyroid hormone (preproPTH) mRNA levels when compared with cells incubated in 1.0 mmol calcium/l, but low calcium concentrations increased the membrane-bound polysomal content of preproPTH mRNA by 200 +/- 16% (mean +/- S.D.). No preproPTH mRNA was detected on free polysomes. Actinomycin D (5 and 10 micrograms/ml) had no effect on steady-state preproPTH mRNA levels measured in dot-blot assays after 24 h, but reduced levels in cells incubated in 1.0 mmol calcium/l to 54 +/- 16% and 39 +/- 12% of control values respectively after 48 h of incubation. Similarly, in cells incubated in 0.4 mmol calcium/l, actinomycin D (5 and 10 micrograms/ml) reduced steady-state preproPTH mRNA levels to 57 +/- 13% and 45 +/- 5% of control values respectively. Actinomycin D did not prevent the rise in polysomal content of preproPTH mRNA induced in cells by incubation in 0.4 mmol calcium/l, but increased polysomal content in cells incubated in 0.4 and 1.0 mmol calcium/l by 159 +/- 9% and 164 +/- 13% respectively after 48 h. These results demonstrate post-transcriptional regulation of PTH synthesis in cultured bovine parathyroid cells, and suggest that this control involves a protein which may be calcium-sensitive.
J Mol Endocrinol 1993 Feb
PMID:Post-transcriptional regulation of bovine parathyroid hormone synthesis. 845 38

The human PTH-related peptide (PTHrP) gene comprises eight exons spanning more than 15 kilobases of genomic DNA. The gene has a highly complex controlling region, which contains four alternatively spliced, noncoding exons and at least two putative promoters, one 5' of exon 1A (up-stream TATA element) and the other 5' of exon 2 (down-stream TATA element). To define important cis regulatory sequences of this gene, a functional dissection of PTHrP 5'-flanking DNA was initiated, using chimeric PTHrP-chloramphenicol acetyltransferase (CAT) constructs. This analysis was carried out in PTHrP-negative human renal carcinoma cells, so that RNA derived from transfected DNA could be studied without interference from endogenous PTHrP sequences. Of the initial series of constructs prepared, the most active was a 1.1-kilobase BamHI-HindIII PTHrP-CAT plasmid containing 350 basepairs of DNA 5' of exon 1C and extending into exon 3. Analysis of transfection products by RNase protection and primer extension revealed that this region contains a previously unrecognized promoter of the gene. This element is located immediately 5' of exon 1C, is active in transfected cells when cloned in isolation up-stream of the CAT gene, and appears to be functional in a number of cell lines and tissues on the basis of primer extension analysis. Unlike the other two PTHrP gene promoters, this element is GC rich and does not possess canonical TATA or CAAT sequences. The human PTHrP gene is one of a handful of genes that appear to use both TATA and GC-rich promoter elements.
Mol Endocrinol 1993 Feb
PMID:Identification and characterization of a GC-rich promoter of the human parathyroid hormone-related peptide gene. 846 40

We examined the effect of PTH on the activity of alpha 1(I) collagen promoter fusion genes in cultured calvariae from transgenic mice. The parent construct, ColCAT 3.6, contains 3520 basepairs of 5' rat alpha 1(I) collagen DNA, 115 basepairs of untranslated alpha 1(I) collagen-coding DNA, and the bacterial chloramphenicol acetyltransferase reporter gene, while the 5'-deletion ColCAT 2.3 contains 2296 kilobases of rat alpha 1(I) collagen promoter sequence. Transgenic mouse lines harboring these collagen promoter fusion genes were developed using the oocyte microinjection technique, and for each construct, three different lines of mice were tested. Calvariae from 6- to 8-day-old transgenic mice were cultured for 48 h with or without bovine PTH-(1-34). ColCAT 3.6 and ColCAT 2.3 were expressed at comparable levels in calvariae and were inhibited by PTH. There were parallel decreases in the incorporation of [3H]proline into collagen and levels of the endogenous alpha 1(I) collagen mRNA and transgene mRNA. Forskolin at 10 microM mimicked the inhibitory effect of PTH on promoter activity in ColCAT 3.6 and ColCAT 2.3 calvariae. A RNase protection assay showed that the transgene was initiated correctly from the transgene promoter. These data show that PTH and cAMP can repress collagen promoter activity in calvariae from transgenic mice, suggesting that the alpha 1(I) collagen promoter may contain cis elements down-stream of -2.3 kilobases that mediate PTH and cAMP repression of collagen gene expression in bone. Cultured bone explants from transgenic mice can be used as a model to study hormonal regulation of alpha 1(I) collagen promoter constructs.
Mol Endocrinol 1993 Mar
PMID:Parathyroid hormone represses alpha 1(I) collagen promoter activity in cultured calvariae from neonatal transgenic mice. 848 79

Calcium-activated neutral protease activity was detected in mouse MC3T3-E1 cell extracts. Inclusion of the cysteine protease inhibitor, E64c, reduced the activity, while pretreatment of intact cells with 10 nM parathyroid hormone for 90 minutes increased it. The presence of calpains in solubilized cells was confirmed by Western blotting using an antibody specific for the 80 K catalytic subunit. These results, combined with the observation that preincubation with a membrane-permeable cysteine protease inhibitor ablates 50% of the PTH-induced osteoblastic retraction, suggest that calpain-catalyzed hydrolysis of regulatory enzymes or structural proteins plays a role in mediating its short-term effects in bone.
Biochem Mol Biol Int 1993 Apr
PMID:Identification of calcium-activated neutral protease activity and regulation by parathyroid hormone in mouse osteoblastic cells. 850 48

To identify determinants in the rat PTH receptor critical for binding the agonist peptide, PTH-(1-34), we systematically replaced 12 segments (5-33 residues) of the receptor's extracellular surface with the corresponding segments of the homologous rat secretin receptor and screened the resulting mutants in COS-7 cells for altered PTH-(1-34) binding properties. Surface expression of mutant receptors was assessed by the binding of monoclonal antibody 12CA5 to the epitope (HA)-tagged receptors. Of the nine well expressed and therefore informative receptor mutants, four bound radiolabeled PTH-(1-34) at levels that were proportional to the corresponding levels of surface expression, whereas five mutants bound [125I]PTH-(1-34) to levels that were lower than predicted from the cell surface expression levels. These five mutations occurred at the extracellular (EC) end of transmembrane domain 1, the carboxy-terminal portion of the first EC loop, the second EC loop, and the third EC loop. We selected for further fine structure analysis the third EC loop; two specific residues, Trp-437 and Gln-440, were identified at which mutations caused 9- to 16-fold reductions in PTH-(1-34)-binding affinity. The same mutations had little or no effect on the binding affinity of PTH-(3-34). This study provides new information on the location of PTH receptor regions important for high affinity agonist binding and identifies two residues in the third extracellular loop which may contribute to interactions involving the hormone's critical amino terminus.
Mol Endocrinol 1995 Oct
PMID:Homolog-scanning mutagenesis of the parathyroid hormone (PTH) receptor reveals PTH-(1-34) binding determinants in the third extracellular loop. 854 35

The PTH/PTH-related peptide (PTHrP) receptor and the calcitonin receptor mediate the action of their physiological ligands by activating two different effectors, adenylyl cyclase and phospholipase C. Whereas regulation of adenylyl cyclase via both receptors is thought to involve the G protein G(s), it is not known whether activation of phospholipase C results from coupling of the receptors to G(q) family members or whether beta gamma-subunit released from receptor-activated G(s) lead to phospholipase C activation. To elucidate the mechanism of this type of dual signaling, we reconstituted the signal transduction of the PTH/PTHrP and the calcitonin receptor in COS-7 and HEK293 cells. In COS-7 cells expressing the receptor alone, addition of the respective ligands resulted in the accumulation of cAMP and inositol phosphates. When cells were cotransfected with the cDNAs of receptor and different alpha-subunits of the Gq family (G alpha q, G alpha 11, G alpha 14, G alpha 15, and G alpha 16, a severalfold increase in the ligand-dependent inositol phosphate production could be observed, indicating that the receptors functionally interacted with all alpha-subunits of the G alpha q family. Additionally, whereas PTH treatment of HEK293 cells coexpressing both the PTH/PTHrP receptor and G alpha q increased both second messengers, the same treatment in cells expressing the PTH/PTHrP receptor alone increased only cAMP. Under all conditions tested, activation of phospholipase C via the PTH/PTHrP and calcitonin receptor required higher ligand concentrations than receptor-mediated adenylyl cyclase activation. Our data strongly support the idea that dual signaling of the PTH/PTHrP and calcitonin receptors is due to the a activation of different G proteins belonging to the G(s) and G(q) families.
Mol Endocrinol 1996 May
PMID:G alpha q family members couple parathyroid hormone (PTH)/PTH-related peptide and calcitonin receptors to phospholipase C in COS-7 cells. 873 87

Due to the importance of Ca2+ in the regulation of vital cellular and tissue functions, the concentration of Ca2+ in body fluids is closely guarded by an efficient feedback control system. This system includes Ca(2+)-transporting subsystems (bone, and kidney), Ca2+ sensing, possibly by a calcium-sensing receptor, and calcium-regulating hormones (parathyroid hormone [PTH], calcitonin [CT], and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]). In humans and birds, acute Ca2+ perturbations are handled mainly by modulation of kidney Ca2+ reabsorption and by bone Ca2+ flow under PTH and possibly CT regulation, respectively. Chronic perturbations are also handled by the more sluggish but economic regulatory action of 1,25(OH2)D3 on intestinal calcium absorption. Peptide hormone secretion is modulated by Ca2+ and several secretagogues. The hormones' signal is produced by interaction with their respective receptors, which evokes the cAMP and phospholipase C-IP3-Ca2+ signal transduction pathways. 1,25 (OH)2D3 operates through a cytoplasmic receptor in controlling transcription and through a membrane receptor that activates the Ca2+ and phospholipase C messenger system. The calciotropic hormones also influence processes not directly associated with Ca2+ regulation, such as cell differentiation, and may thus affect the calcium-regulating subsystems also indirectly.
Crit Rev Biochem Mol Biol 1996 Feb
PMID:Homeostatic control of plasma calcium concentration. 874 55

Expression of the gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is down-regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Deletions of the 5'-flanking region of the rat PTHrP gene, when fused to the chloramphenicol acetyl-transferase gene and transfected into ROS 17/2.8 (rat osteosarcoma) cells, showed that the 1,25-(OH)2D3 responsive region is located between -1.05 and -0.71 kb upstream of the transcription start site. Further mapping of this region revealed that a 123-bp fragment is able to confer 1,25-(OH)2D3 responsiveness to a heterologous (SV40) promoter. This region contains two potential vitamin D response elements (VDREs). One of these motifs resembles the negative VDRE (nVDRE) from the PTH gene, which is also down-regulated by vitamin D3. The other element resembles the canonical VDRE (two hexanucleotide motifs separated by three nucleotides), which has been characterized in a number of genes whose expression is modulated by vitamin D3. Electrophoretic mobility shift assays using nuclear extracts from ROS 17/2,8 cells and from vitamin D receptor. (VDR)-enriched COS 1 cells revealed that both elements interact with the VDR. This protein-DNA interaction is disrupted by an anti-VDR antibody. Therefore, modulation of PTHrP gene transcription by 1,25-(OH)2D3 is mediated by the VDR interacting with one or both of the identified motifs in the 5'-flanking sequence of the gene.
Mol Endocrinol 1996 Jun
PMID:DNA sequences in the rat parathyroid hormone-related peptide gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression. 877 27

Interstitial collagenase is secreted by the osteoblast in response to bone-resorbing agents. Previously, we cloned the rat interstitial collagenase cDNA from UMR 106-01 rat osteoblastic osteosarcoma cells. We demonstrated that induction of collagenase by PTH, a powerful resorbing agent, in UMR 106-01 cells is in part transcriptional. In the present study we isolate and characterize the rat interstitial collagenase gene. The gene consists of 10 exons and spans approximately 12 kbp. The major transcriptional start site, determined by primer extension analysis and confirmed by RNase protection assay, is 25 nucleotides upstream of the translational start site. The previously isolated cDNA was missing the 5'-untranslated sequence in addition to 17 nucleotides of the signal sequence of the preproenzyme; therefore, we also present these data. Chloramphenicol acetyl transferase (CAT) analyses were performed on the 5'-upstream region of the gene. These data indicate that PTH appears to mediate its effect through an AP-1 consensus-binding sequence (-51). Footprint analysis demonstrates protein binding to this site. Site-specific mutagenesis markedly decreased protein binding, which correlated directly with a decrease in CAT activation by PTH. Supershift data indicate that cAMP response element binding protein (CREB) is binding to this AP-1 consensus sequence. In addition we demonstrate that PTH induces phosphorylation of CREB.
Mol Endocrinol 1996 Jul
PMID:Parathyroid hormone induction of rat interstitial collagenase mRNA in osteosarcoma cells is mediated through an AP-1-binding site. 881 27


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