Plasminogen activator regulation in osteoblasts: parathyroid hormone inhibition of type-1 plasminogen activator inhibitor and its mRNA.

In order to determine the mechanism by which parathyroid hormone (PTH) stimulates plasminogen activator (PA) activity in rat osteoblasts, we investigated the effect of human PTH(1-34) [hPTH(1-34)] on the synthesis of mRNAs for tissue-type PA (tPA), urokinase-type PA (uPA), and PA inhibitor-1 (PAI-1), and on release of PA activity and PAI-1 protein in both normal rat calvarial osteoblasts and UMR 106-01 osteogenic sarcoma cells. hPTH(1-34) (0.25-25 nM) decreased PAI-1 mRNA and protein, and increased PA activity in both cell types in a dose-dependent manner with ED50 of about 1 nM for both responses. Forskolin and isobutylmethylxanthine also stimulated PA activity and decreased PAI-1 protein and mRNA in both cell types. hPTH(1-34) did not show any consistent effect on tPA and uPA mRNA in calvarial osteoblasts, but a modest (two-fold) increase of both mRNAs was observed in UMR 106-01 cells treated with 25 nM hPTH(1-34). However, when protein synthesis was inhibited with 100 microM cycloheximide, the increase of tPA and uPA mRNA by hPTH(1-34) was enhanced in UMR 106-01 cells and became evident in calvarial osteoblasts. Fibrin autography also revealed that hPTH(1-34) increases tPA and uPA activity, especially after cycloheximide treatment in UMR 106-01 cells. These results strongly suggest that PTH increases PA activity predominantly by decreasing PAI-1 protein production through a cyclic adenosine monophosphate (cAMP)-dependent mechanism in rat osteoblasts. The reduction of PAI-1 protein by PTH results in enhanced action of both tPA and uPA, and would contribute to the specific roles of these PAs in bone.     

Rat RI beta isoform of type I regulatory subunit of cyclic adenosine monophosphate-dependent protein kinase: cDNA sequence analysis, mRNA tissue specificity, and rat/mouse difference in expression in testis

A rat complementary DNA (cDNA) for the RI beta isoform of type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3'-untranslated regions, with a single (ATTAAA) poly-adenylation site. The largest open reading frame was preceded by a short out-of-phase open reading frame, which is not seen in the corresponding mouse RI beta cDNA due to a single base substitution. The rat RI beta cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RI beta mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RI beta cDNA also detected RI beta mRNA in mouse brain, but not mouse testis, from 10-week-old BALB/c or 10- and 6-week-old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RI beta in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RI beta mRNA.     

Parathyroid hormone stimulation and PKA signaling of latent transforming growth factor-beta binding protein-1 (LTBP-1) mRNA expression in osteoblastic cells

Parathyroid hormone (PTH) regulates bone remodeling and calcium homeostasis by acting on osteoblasts. Recently, the gene expression profile changes in the rat PTH (1-34, 10(-8)M)-treated rat osteoblastic osteosarcoma cell line, UMR 106-01, using DNA microarray analysis showed that mRNA for LTBP-1, a latent transforming growth factor (TGF-beta)-binding protein is stimulated by PTH. Latent TGF-beta binding proteins (LTBPs) are required for the proper folding and secretion of TGF-beta, thus modifying the activity of TGF-beta, which is a local factor necessary for bone remodeling. We show here by real time RT-PCR that PTH-stimulated LTBP-1 mRNA expression in rat and mouse preosteoblastic cells. PTH also stimulated LTBP-1 mRNA expression in all stages of rat primary osteoblastic cells but extended expression was found in differentiating osteoblasts. PTH also stimulated TGF-beta1 mRNA expression in rat primary osteoblastic cells, indicating a link between systemic and local factors for intracellular signaling in osteoblasts. An additive effect on LTBP-1 mRNA expression was found when UMR 106-01 cells were treated with PTH and TGF-beta1 together. We further examined the signaling pathways responsible for PTH-stimulated LTBP-1 and TGF-beta1 mRNA expression in UMR 106-01 cells. The PTH stimulation of LTBP-1 and TGF-beta1 mRNA expression was dependent on the PKA and the MAPK (MEK and p38 MAPK) pathways, respectively in these cells, suggesting that PTH mediates its effects on osteoblasts by several intracellular signaling pathways. Overall, we demonstrate here that PTH stimulates LTBP-1 mRNA expression in osteoblastic cells and this is PKA-dependent. This event may be important for PTH action via TGF-beta in bone remodeling.     

Isolation and sequence of cDNA clones encoding rat phosphatidylinositol transfer protein

Phosphatidylinositol (PtdIns) transfer protein is a cytosolic protein that catalyzes the transfer of PtdIns between membranes. It is expressed in organisms from yeast to man, and activity has been found in all animal tissues examined. Using antibodies prepared against bovine brain PtdIns transfer protein, lambda gt11 rat brain cDNA libraries were screened and several clones isolated. DNA sequence analysis showed that the cDNAs encoded a polypeptide of 271 amino acids with a mass of 31,911 Da. Comparison of the deduced amino acid sequence with N-terminal sequence data obtained for the intact purified bovine brain protein and rat lung phospholipid transfer protein verified that the cDNAs were PtdIns transfer protein clones. The predicted protein shows no significant sequence similarity to other known (phospholipid)-binding proteins. DNA blot hybridization suggests that the rat genome may contain more than one gene encoding PtdIns transfer protein. RNA blot hybridization reveals that the PtdIns transfer protein gene is expressed at low levels in a wide variety of rat tissues; all tissues examined showed a major mRNA component of 1.9 kilobases and a minor component of 3.4 kilobases. The isolation of clones encoding rat PtdIns transfer protein will greatly facilitate studies of the structure and function of PtdIns transfer proteins and their role in lipid metabolism.     

Up-regulation of connexin 43 and gap junctional intercellular communication by Coleusin Factor is associated with growth inhibition in rat osteosarcoma UMR106 cells

Gap junctions, formed by connexin (Cx) family proteins, permit direct exchange of regulatory ions and small signal molecules between neighbouring cells. Gap junctional intercellular communication (GJIC) plays an important role in maintaining the homeostasis and preventing cell transformation. Most of the tumour cells feature deficient or aberrant connexin expression and GJIC level, and restoration of connexin expression and GJIC is correlated with cell growth control. Numerous researches has suggested the possibility of connexins as potential anti-tumour targets for chemoprevention and chemotherapy. We investigated the ability of Coleusin Factor (CF, also named FSK88) to regulate the Cx43 expression and GJIC level in rat osteosarcoma UMR106 cells. The results have demonstrated that CF increased the mRNA and protein expression of Cx43 in both in a dose- and timedependent manner, and concomitant with up-regulation of Cx43, CF treatment up-regulated the diminished GJIC level in UMR106 cells as assayed by dye transfer experiments. In addition, Cx43 distribution at the plasma membrane was also enhanced dramatically by CF treatment. Furthermore, we discovered that CF was potent to inhibit the growth and proliferation of UMR106 cells. These results provide the first evidence that CF can regulate connexin and GJIC, indicating that Cx43 may be a target of CF to exert its anti-tumour effects.     

Molecular cloning, cDNA structure and tissue-specific expression of the human regulatory subunit RI beta of cAMP-dependent protein kinases.

Complementary DNA clones for the regulatory subunit RI beta of cAMP-dependent protein kinases were isolated from a human testis cDNA library using a mouse RI beta cDNA probe. One clone 2.4 kilobases (kb) in length contained an open reading frame of 1137 bases, and encoded a protein of 379 amino acids (excluding the initiator methionine). The human RI beta protein was one amino acid shorter than the corresponding protein in mouse and rat. The nucleotide similarity to mouse and rat sequences was 85.6% and 84.8%, respectively, while the amino acid similarity was 97.6% and 97.3%, respectively. Northern blot analyses revealed a 2.7 kb mRNA in human tissues and a 2.8 kb mRNA in mouse tissues. Both mouse and human RI beta mRNA were found to be expressed in most tissues, and not restricted to brain and testis as reported by others.     

Osteoblasts display receptors for and responses to leukemia-inhibitory factor

Specific binding of leukemia-inhibitory factor (LIF) to osteoblasts, but not multinucleated osteoclasts, was demonstrated by receptor autoradiography by using cells isolated from newborn rat long bones. The clonal rat osteogenic sarcoma cells, UMR 106-06, which have several phenotypic properties of osteoblasts, expressed 300 LIF receptors per cell, with an apparent KD of 60 pM. Treatment of calvarial osteoblasts or UMR 106-01 cells with LIF resulted in a dose-dependent inhibition of plasminogen activator (PA) activity. Both calvarial osteoblasts and osteogenic sarcoma cells were shown by Western blotting and reverse fibrin autography to produce plasminogen activator inhibitor-1 (PAI-1), the production of which was increased by LIF treatment. Northern blot analysis revealed that LIF treatment resulted in a rapid (peak 1 hour), dose-dependent increase in mRNA for PAI-1. LIF treatment of the preosteoblast cell line, UMR 201, enhanced the alkaline phosphatase response of these cells to retinoic acid. Each of the osteoblast-like cell types (calvarial osteoblasts, UMR 106-06, and UMR 201) was shown to produce LIF by bioassay and, by using the polymerase chain reaction (PCR), was shown to express low levels of mRNA for LIF. These data establish that cells of the osteoblast lineage are targets for LIF action. The reported anabolic effects of this cytokine on bone formation in vivo could be related to inhibition of protease activity. LIF may be an important paracrine modulator in bone, or perhaps an autocrine one, based on the evidence for its production by osteoblasts and osteoblast-like cells.     

RFP_（134）对UMR106细胞EGF受体酪氨酸蛋白激酶的调节作用

以大鼠成骨肉瘤细胞（ＵＭＲ１０６）为模型,研究了表皮生长因子（ＥＧＦ）对其受体酪氨酸蛋白激酶（ＴＰＫ）的调节作用。以本实验室从植物中提取纯化的二萜类活性物质（ＲＦＰ１３４）为诱导分化剂,观察了ＲＦＰ１３４对ＵＭＲ１０６细胞ＥＧＦ受体ＴＰＫ的活性和磷酸化作用的影响,并与ＲＡ和ＲＦＰ１３４＋ＲＡ处理细胞做了比较,结果显示ＥＧＦ与其受体结合后能激活ＴＰＫ,使ＴＰＫ活性增加２倍．ＲＦＰ１３４,ＲＡ,ＲＦＰ１３４＋ＲＡ处理细胞后,分别降低ＥＧＦ诱导的受体ＴＰＫ活性５０％,４３％,５５％,降低磷酸化ＴＰＫ含量５５％,３６％,５３％。从结果中发现无ＥＧＦ刺激的细胞也具有受体ＴＰＫ磷酸化作用,用ＲＦＰ１３４,ＲＡ,ＲＦＰ１３４＋ＲＡ处理细胞,分别降低受体磷酸化ＴＰＫ含量５９％,４０％,５７％,而且我们发现用ＥＧＦ诱导的细胞受体ＴＰＫ含量高于无ＥＧＦ作用的细胞．提示ＵＭＲ１０６细胞本身可能具有受体ＴＰＫ活性,能够引起细胞受体自动磷酸化,ＥＧＦ刺激后ＴＰＫ的磷酸化作用增强,可见ＲＦＰ１３４对ＥＧＦ诱导的ＴＰＫ磷酸化和无ＥＧＦ诱导的受体自动磷酸化都具有明显的抑制作用,（并强于ＲＡ）这可能与在第二信使水平上阻抑ＰＴＰＫ活性密切相关     

Temporal gradients in shear stimulate osteoblastic proliferation via ERK1/2 and retinoblastoma protein

Bone cells are subject to interstitial fluid flow (IFF) driven by venous pressure and mechanical loading. Rapid dynamic changes in mechanical loading cause transient gradients in IFF. The effects of pulsatile flow (temporal gradients in fluid shear) on rat UMR106 cells and rat primary osteoblastic cells were studied. Pulsatile flow induced a 95% increase in S-phase UMR106 cells compared with static controls. In contrast, ramped steady flow stimulated only a 3% increase. Similar patterns of S-phase induction were also observed in rat primary osteoblastic cells. Pulsatile flow significantly increased relative UMR106 cell number by 37 and 62% at 1.5 and 24 h, respectively. Pulsatile flow also significantly increased extracellular signal-regulated kinase (ERK1/2) phosphorylation by 418%, whereas ramped steady flow reduced ERK1/2 activation to 17% of control. Correspondingly, retinoblastoma protein was significantly phosphorylated by pulsatile fluid flow. Inhibition of mitogen-activated protein (MAP)/ERK kinase (MEK)1/2 by U0126 (a specific MEK1/2 inhibitor) reduced shear-induced ERK1/2 phosphorylation and cell proliferation. These findings suggest that temporal gradients in fluid shear stress are potent stimuli of bone cell proliferation.     

Molecular cloning of rat insulin-like growth factor I complementary deoxyribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues

Two classes of insulin-like growth factor I (IGF-I) cDNAs were isolated from an adult rat liver library using a human IGF-I cDNA probe. The two types of rat IGF-I cDNA differed by the presence or absence of a 52-base pair insert which altered the derived C-terminal amino acid sequence of the E peptide, but not the 3'-untranslated region or the sequence coding for the mature IGF-I protein. When probes derived from these cDNA clones were hybridized to Northern blots of rat mRNA, specific bands of 8.6, 2.1, and 1.0-1.4 kilobases were seen. Hybridization to poly(A)+ RNA from various tissues from GH-treated and control rats demonstrated an increase in IGF-I mRNA due to GH treatment in all tissues examined.     

Expression of adenylate cyclase-coupled osseous parathyroid hormone and parathyroid hormone-like peptide receptors in Xenopus oocytes.

Functional parathyroid hormone (PTH) and PTH-like peptide receptors were expressed in Xenopus laevis oocytes after injection of poly(A)+ RNA isolated from the rat osteogenic sarcoma cell line, UMR 106. Increases in cAMP were seen in individual oocytes in response to added bovine (b) PTH-(1-34) (10(-6) M), human (h) PLP-(1-34) (hPLP-(1-34), 10(-6) M), isoproterenol (10(-4) M), and forskolin (10(-4) M). Although both intracellular and extracellular cAMP levels were stimulated approximately 1.5-2-fold by these agonists, intracellular concentrations of cAMP were substantially higher than extracellular concentrations. Peak increases with bPTH-(1-34) occurred after a 30-min incubation with the hormone 48 h after oocyte injection. bPTH-(1-34) caused a concentration-dependent augmentation of cAMP in injected oocytes, and the in vitro antagonist hPLP-(3-34) produced dose-dependent inhibition of both bPTH-(1-34)- and hPLP-(1-34)-stimulated cAMP accumulation. Specific binding of PTH to oocyte membranes was also demonstrated 48 h after oocyte injection with UMR 106 cell mRNA. Following size fractionation of isolated UMR 106 poly(A)+ RNA by sucrose density gradients, mRNA directing the expression of both PTH- and PLP-stimulated cAMP in oocytes appeared in the 3.5-4.9-kilobase fraction. These results demonstrate that adenylate cyclase-coupled osseous PTH and PLP receptors can be expressed after injection of naturally occurring mRNA into Xenopus oocytes, that PTH- and PLP-stimulated increases in cAMP concentrations can be detected in individual oocytes injected with bone cell-derived mRNA, that PTH and PLP appear to cross-react at a common receptor after injection of UMR 106 cell mRNA into oocytes, and that size selection of mRNA encoding the PTH and PLP receptors can be achieved by density gradient centrifugation. These studies, therefore, indicate the potential usefulness of the Xenopus oocyte system in expression cloning of PTH and PLP receptor cDNAs and illustrate the feasibility of employing this system to examine the biology of PTH and PLP receptors.     

1,25-Dihydroxyvitamin D3 stimulates the synthesis of matrix gamma-carboxyglutamic acid protein by osteosarcoma cells. Mutually exclusive expression of vitamin K-dependent bone proteins by clonal osteoblastic cell lines

Several clonal rat osteosarcoma cell lines were tested for the ability to express and secrete matrix Gla protein (MGP), a small vitamin K-dependent protein found in bone and cartilage. Two independently derived cell lines, UMR 106-01 and ROS 25/1, expressed MGP mRNA and secreted MGP antigen identical in size with that found in bone. No MGP message could be detected in ROS 17/2 and 2/3 cells, cell lines previously shown to synthesize the other known vitamin K-dependent bone protein, bone Gla protein (BGP), and no BGP mRNA could be detected in the cell lines which synthesize MGP. Since UMR 106-01 and ROS 17/2 are presently the best characterized clonal osteoblastic cell lines, the discovery of the mutually exclusive expression of MGP and BGP by these cell lines indicates that osteosarcoma cells can be fixed in different phenotypic states and that MGP and BGP should be useful markers for the analysis of phenotypic expression in bone. Treatment of UMR 106-01 cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) dramatically increased MGP mRNA within 4 h and, by 24 h, increased MGP secretion 15-fold. This is only the second example of a bone matrix protein whose synthesis is dramatically increased by vitamin D, the first being the 6-fold stimulation of BGP synthesis by 1,25(OH)2D3 in ROS 17/2 cells. The discovery that MGP and BGP are similarily regulated by 1,25(OH)2D3 was unexpected since the two proteins differ markedly in structure, physical properties, and tissue distribution. Since the synthesis of MGP is rapidly and dramatically increased by 1,25(OH)2D3, it is probable that MGP plays a role in the normal bone response to the hormone. MGP may also be the vitamin K-dependent protein whose abnormal synthesis in the Warfarin-treated animal modifies the bone response to 1,25(OH)2D3.