Cortisol Decreases Hepatocyte Growth Factor Levels in Human Osteoblast-Like Cells

Osteoporosis is a well-known side effect of long-term treatment with glucocorticoids. The hepatocyte growth factor (HGF) receptor is expressed by human osteoclasts and osteoblasts, and mouse osteoblasts also express HGF, indicating that HGF may regulate bone metabolism. Because HGF could be a candidate factor in the local paracrine signaling between osteoblasts and osteoclasts in bone, we decided to study whether human osteoblasts secrete HGF and whether glucocorticoids regulate the expression of HGF. HGF was easily detectable in the culture medium from human osteoblast-like cells (hOB). The HGF protein released into the culture medium was increased with increasing confluency. Hydrocortisone decreased the amount of HGF released into the culture medium from hOB in a dose-dependent manner with a maximal effect at 10⁻ M. Time-course studies revealed that hydrocortisone decreased the amount of HGF released into the culture medium significantly after 16 hours of stimulation (65 ± 2% of control culture). This effect of hydrocortisone was maximal after 24 hours of stimulation (52 ± 8% of control culture). In conclusion, HGF is produced by primary cultured hOB cells. Furthermore, the amount of HGF released into the culture medium is decreased by glucocorticoids. The biological significance of this finding remains to be demonstrated. Received: 29 December 1998 / Accepted: 13 August 1999     

Osteogenic Protein-1 Stimulates mRNA Levels of BMP-6 and Decreases mRNA Levels of BMP-2 and -4 in Human Osteosarcoma Cells

Bone morphogenetic proteins (BMPs) are novel growth and differentiation factors that act on mesenchymal stem cells to initiate new bone formation in vivo and promote the growth and differentiation of cells in the osteoblastic lineage. In the present study, we examined the effects of recombinant human osteogenic protein-1 (also known as BMP-7) on the expression of related members of the BMP family using SaOS-2 and U2-OS, two human osteosarcoma cell strains. Evaluation of BMP-2, -4, and -6 mRNA expression indicates that OP-1 stimulated the mRNA levels of BMP-6 in both SaOS-2 cells (threefold) and U2-OS cells (fivefold) after 24 hours of treatment, while decreasing the mRNA levels of BMP-4 in SaOS-2 cells (80%) and BMP-2 and BMP-4 in U2-OS cells by 50% and 72%, respectively. BMP-2 mRNA expression, as examined by Northern blot analysis, was below detectable limits in SaOS-2 cultures. These results demonstrate that OP-1 modulates the mRNA expression of related members of the BMP family, suggesting a possible mode of action of OP-1 on the growth and differentiation of cells in the osteoblastic lineage in vitro. Received: 7 May 1996 / Accepted: 24 September 1996     

TGFβ-1 mRNA Expression and Proliferation of Human Osteoblastic Cells in Nonosteoporotic and Osteoporotic Women under Influence of TGFβ-1 and IGF-I

Currently, primary osteoporosis is the most frequent metabolic disease in women after menopause [1]. The resulting loss of bone mass is accompanied by an increased risk of skeletal fragility. One reason for the development of osteoporosis might be an impaired function of mature osteoblasts. To evaluate the involvement of specific growth factors in bone remodeling, cell cultures of osteoblastic cells derived from nonosteoporotic and osteoporotic postmenopausal women were established. The influences of TGFβ-1 and IGF-I on proliferation and mRNA expression of TGFβ-1 were investigated by [³H]-thymidine incorporation and competitive RT-PCR. We found IGF-I to have no significant effect on proliferation in cells of osteoporotic and nonosteoporotic patients. In contrast, differences were found in TGFβ-1 mRNA expression after application of IGF-I. Application of TGFβ-1 enhanced its own mRNA expression in both groups in a similar manner. Whereas the proliferation of cells of nonosteoporotic patients was inhibited by (10⁻¹⁰ M) TGFβ-1, this treatment led to an increased proliferation of cells of osteoporotic patients. Received: 10 June 1996 / Accepted: 13 January 1997     

Down-Regulation of Osteoblastic Cell Differentiation by Epidermal Growth Factor Receptor

The role of epidermal growth factor receptors (EGF-R) in osteogenic cell differentiation was investigated using preosteoblastic MC3T3-E1 (MC3T3) cells and osteoblast-like ROS 17/2.8 (ROS) cells. When cultured in the presence of β-glycerophosphate (GP) and ascorbic acid (AA), MC3T3 cells underwent spontaneous differentiation into osteoblasts which was confirmed as they expressed osteoblast markers such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). Interestingly, the number of EGF-binding sites decreased during their differentiation into osteoblasts, and the osteogenic protein-1 (OP-1) treatment, which accelerated their differentiation, lowered the number of EGF-binding sites even further. On the other hand, ROS cells with high expression levels of osteoblast markers and no EGF-R, after being transfected with human EGF-R cDNA (EROS cells), expressed numerous EGF-binding sites as well as EGF-R mRNA and protein; in the process, they ceased to express osteoblast markers, indicating their dedifferentiation into osteoprogenitor cells. Both MC3T3 and EROS cells showed increased cell growth in response to EGF, whereas ROS cells did not. These results imply that the EGF/EGF-R system in osteogenic cells has a crucial function in osteoblast phenotype suppression and osteogenic cell proliferation.     

Regulation of c-fos and c-jun Expression by Calcitonin in Human Breast Cancer Cells

Breast cancer cells (BCC) have calcitonin (CT) receptors, yet the action of the hormone on these cells is largely unknown. We found that CT produced a strong and transient time- and dose-dependent increase in c-fos mRNA in BCC lines. This event was prevented by a protein kinase A (PKA) inhibitor, H89. CT alone did not influence the expression of c-jun and of the tissue inhibitors of metalloproteases (timp) -1 and -2 mRNAs; however, it reduced the induction of these mRNAs by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), without apparent changes in the half-life of the mRNA (measured for c-jun). Along the same line, CT reduced the c-jun induction and T-47D growth stimulation by epidermal growth factor (EGF) and insulin. These effects were mimicked by forskolin and/or prevented by H89, suggesting that PKA activation was involved. These results indicate that CT modulates in BCC the mRNA levels of two important growth-related early response genes (c-fos and c-jun) and of two other genes (timp-1 and -2) involved in the control of metastatic events. Received: 2 May 1996 / Accepted: 10 December 1996     

Basic Fibroblast Growth Factor in the Presence of Dexamethasone Stimulates Colony Formation, Expansion, and Osteoblastic Differentiation by Rat Bone Marrow Stromal Cells

Basic fibroblast growth factor (bFGF) is known to stimulate endosteal bone formation in vivo by a mechanism possibly mediated via osteoblast precursor cells present in the bone marrow. In high density cultures of primary bone marrow cells, and in the presence of glucocorticoids, bFGF stimulates the formation of a bone-like matrix; however, due to the dense nature of these cultures, the exact mechanism of action is unclear. In an adaptation of the fibroblastic colony formation unit assay, in which the bone marrow cells are grown in the presence of dexamethasone, β-glycerophosphate, and ascorbate, mineralized colonies are formed which stem from single mesenchymal precursor cells and grow in isolation from each other. Using this system we have been able to investigate the mechanism by which bFGF stimulates the formation of bone like tissue in vitro. We have shown that bFGF increases the formation of a calcified collagenous matrix in vitro by (1) increasing the total number of fibroblastic colonies formed, (2) increasing the proportion of differentiated colonies that synthesize collagen and calcify, and (3) stimulating the proliferation and collagen accumulation of the individual colonies. A maximal increase in total and differentiated colony numbers was seen after only 5 days exposure to bFGF, however, continued exposure to bFGF continued to increase the size and collagen content of the individual colonies. Bearing in mind the endosteal location of newly formed bone seen after treatment with bFGF, these processes may well play an active role in this effect. Received: 17 January 1997 / Accepted: 30 July 1998     

Hormonal Regulation of the Osteoblastic Phenotype Expression in Neonatal Murine Calvarial Cells

Osteoblastic cell cultures from fetal rat calvariae have provided a popular model for studying the effects of dexamethasone (DEX) and 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] on gene expression but data from murine calvarial cells are scarce. Species-specific responses of rat and mouse osteoblastic cells to these hormones have been reported previously. In the present study, we investigated the effects of DEX and 1,25(OH)₂D₃ on expression of the osteoblastic phenotype by mouse calvarial cells. These murine osteoblast-like (MOB) cells expressed alkaline phosphatase (ALP) activity and osteocalcin and formed calcified nodules. Unlike the rat calvarial cells, ALP activities and nodule formation in MOB were inhibited by DEX. 1,25(OH)₂D₃ enhanced and DEX lowered the amount of osteocalcin synthesized by MOB. 1,25(OH)₂D₃ did not affect the number of nodules, but increased their sizes. Treating the cells for 2 days with only DEX at the beginning of the culture enhanced the effect of 1,25(OH)₂D₃ on ALP. We found that in murine calvarial cells, DEX inhibits and 1,25(OH)₂D₃ enhances ALP activity, osteocalcin synthesis, and calcified nodule formation. This is in contrast to previous reports of rat calvarial cells where DEX is a positive and 1,25(OH)₂D₃ can be a negative regulator of the osteoblastic phenotype. These results suggest that profound species-specific differences exist between mice and rats in the regulation of the osteoblastic phenotype. Received: 15 October 1997 / Accepted: 16 June 1998     

Ionized Calcium and Bone Turnover in the Estrogen-Deficient Rat

Our knowledge of the concentration of growth factors in growing bone is limited. In the present study, we examined the developmental changes in the concentrations of insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-β) in the rat femur between weanling and maturity. We show that during the rapid growth phase there is a continuous rise in bone matrix IGF-I and TGF-β in all compartments of the femoral bone. The association between IGF-I and TGF-β is not only temporal, but with few exceptions is also observed within the animals of each age class. These data support the hypothesis that IGF-I and TGF-β play an important role in the growth-associated accumulation of bone mass. Received: 16 April 1997 / Accepted: 9 July 1998     

转化生长因子β1基因产物在人非小细胞肺癌中的表达研究

目的探讨转化生长因子β１基因表达产物在人非小细胞肺癌中的表达水平及其临床意义。方法采用免疫组织化学方法对７３例人非小细胞肺癌组织中转化生长因子β１基因表达蛋白水平进行了研究。结果转化生长因子β１基因表达水平与肺癌的病期、组织学类型、细胞分化程度，以及肺癌转移有密切关系。转化生长因子β１基因表达水平在腺癌明显高于鳞癌和腺鳞癌，低分化癌明显低于中－高分化癌，Ⅲ、Ⅳ期癌明显低于Ⅰ、Ⅱ期癌，转移癌明显低于原发癌，有淋巴结转移的原发癌明显低于无淋巴结转移的原发癌（Ｐ＜０．０１或Ｐ＜０．０５）。结论转化生长因子β１基因参与调控肺癌的发生、发展和转移过程。     

A Human Homolog of the 150 kD Avian Osteoclast Membrane Antigen Related to Superoxide Dismutase and Essential for Bone Resorption is Induced by Developmental Agents and Opposed by Estrogen in FLG 29.1 Cells

Osteoclast development from hematopoietic bone marrow precursors is associated with the expression of various enzymes, receptors, adhesion molecules, and other specialized components. Among these is a novel 150 kD superoxide dismutase-related membrane glycoprotein, originally identified by its reaction with the anti-osteoclast monoclonal antibody 121F. This antigen is uniquely restricted to osteoclasts in bone, universally present on osteoclasts from multiple species, induced during osteoclast differentiation in vitro and in ovo, and required at high levels for avian osteoclastic bone pit resorption. Expression of a comparable human antigen was investigated using human leukemic FLG 29.1 cells capable of differentiating towards an osteoclast-like phenotype. Phorbol ester, 1,25 (OH)₂ vitamin D₃, and osteoblast-derived soluble factors elicited dose and time-dependent inductions of this antigen as measured by enzyme-linked immunosorbent assay (ELISA) and immunocytochemical staining, coincident with their display of multiple other osteoclastic features. Synergistic interactions of these modulators led to further elevations in the ultimate expression levels of this antigen, although not to the full extent associated with in vivo-formed avian osteoclasts. The potent antiresorptive hormone 17β-estradiol, but not its inactive α isomer, partially suppressed the phorbol ester-induced elevation of the 121F antibody-reactive antigen in FLG 29.1 cells as it does in avian osteoclast-like cells. Characterization of the human antigen isolated from FLG 29.1 cells by 121F immunoaffinity purification demonstrated that this regulated membrane component was synthesized by these human cells, more abundant following their differentiation into osteoclast-like cells, and similar biochemically and immunologically to the 150 kD integral membrane glycoprotein previously described from avian osteoclasts. Therefore, this report is the first documentation that human osteoclast-like FLG 29.1 cells express, in a developmentally regulated fashion, a homolog of the specific 150 kD avian osteoclast surface antigen that is related to superoxide dismutase, a protective free radical scavenging enzyme and is essential for osteoclastic bone resorption. Received: 1 April 1996 / Accepted: 19 July 1996     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998     

Prostaglandin E2 (PGE2) Autoamplifies its Production Through EP1 Subtype of PGE Receptor in Mouse Osteoblastic MC3T3-E1 Cells

Prostaglandin E₂ (PGE₂) is known to autoamplify its production in the osteoblasts through the induction of prostaglandin G/H synthase-2 (PGHS-2), which is the inducible form of the rate-limiting enzyme in PG synthesis, PGHS. To elucidate the cellular mechanism mediating this process, we have employed the PGE₂ analogs, which are specific agonists for four subtypes of PGE receptor, and studied the potency of these analogs to induce PGHS-2 mRNA in mouse osteoblastic MC3T3-E1 cells. The induction was mainly observed by 17-phenyl-ω-trinor PGE₂ (EP₁ agonist) and sulprostone (EP₃/EP₁ agonist), but not by butaprost (EP₂ agonist) or 11-deoxy PGE₁ (EP₄/EP₂ agonist). Since EP₃ subtype was undetectable in MC3T3-E1 cells, these data indicate that PGHS-2 mRNA induction is mediated through EP₁ subtype of PGE receptor in MC3T3-E1 cells. PGE₂ production determined by radioimmunoassay was also increased by 17-phenyl-ω-trinor PGE₂ and sulprostone. The autoamplification of PGE₂ production is considered to be important in elongating the otherwise short-lived PGE₂ action in certain physiological conditions such as mechanical stress and fracture healing, as well as the pathological inflammatory bone loss. The observations in the present study provide us with the better understanding of these processes. Received: 29 April 1997 / Accepted: 22 August 1997     

Age-Associated Decline in Human Femoral Neck Cortical and Trabecular Content of Insulin-Like Growth Factor I: Potential Implications for Age-Related (Type II) Osteoporotic Fracture Occurrence

Recent evidence suggests that regulatory peptides such as insulin-like growth factor-I (IGF-I) are released locally from bone during resorption, and may then act in a sequential manner to regulate the cellular events required for the coupling of bone formation to resorption. Among other factors, a decrease in bone-associated IGF-I levels could therefore result in remodeling imbalance and contribute to the gradual loss of bone that occurs with age. As the femoral neck region is of primary concern for the clinical manifestations of osteoporosis, the current study was intended to assess the IGF-I contents in femoral neck cortical and trabecular bone from aging individuals. Bone samples from the neck region were obtained at postmortem from 39 females and 35 males, aged 23–92 years. Concentrations of IGF-I and osteocalcin were measured by radioimmunoassay in the supernatants obtained after EDTA and guanidine hydrochloride extraction. The total amount of protein present in the extracts was determined by spectrophotometry. IGF-I levels were significantly lower in trabecular compared with cortical bone. Though femoral neck total protein did not vary with donor age, both IGF-I and osteocalcin were found to decline markedly. Between the ages of 23 and 92 years, average yearly rates of loss of 0.30 and 0.21 ng IGF-I/mg protein were observed in cortical and trabecular bone, respectively, corresponding with net losses of nearly 35% of the cortical skeletal content of IGF-I and 41% of the trabecular skeletal content of IGF-I. These changes in bone-associated IGF-I paralleled those of osteocalcin, consistent with an overall decrease in osteoblast function with aging. In women, the rate of decline was significantly faster for trabecular than for cortical IGF-I, however in men, age-dependent changes in cortical and trabecular IGF-I were similar. These findings support the hypothesis that changes in the local IGF regulatory system over time could be a pathophysiologic component of the age-related (type II) femoral neck osteoporotic syndrome. Received: 12 December 1996 / Accepted: 23 April 1997     

Stimulation of Femoral Trabecular Bone Growth in Ovariectomized Rats by Human Parathyroid Hormone (hPTH)-(1-30)NH2

It has been proposed that intermittent bursts of adenylyl cyclase and the surges of cyclic AMP (cAMP) they produce can trigger PTH's bone anabolic action without the activation of phospholipase-C (PLC). This was based on the osteogenic action in ovariectomized (OVX) rats of hPTH-(1-31)NH₂, which can stimulate adenylyl cyclase but not PLC in ROS 17/2 rat osteosarcoma cells, and the osteogenic impotence of fragments such as 1-desamino-hPTH-(1-34) and hPTH-(8-84) which strongly stimulate PLC but not adenylyl cyclase. But this seems to have been disproven by the inability of hPTH-(1-30)NH₂ to stimulate bone growth despite its having hPTH-(1-31)NH₂'s ability to strongly stimulate adenylyl cyclase but not PLC in cells with rat type1 PTH/PTHrP receptors. Because of the importance of hPTH-(1-30)NH₂'s apparent osteogenic impotence for knowing how PTH triggers bone growth, we have reinvestigated the fragment's ability to stimulate trabecular bone growth in the femurs of young OVX rats and have found it to be strongly osteogenic at doses 2–10 times higher than the highest dose used previously. Thus, 6 weeks of once-daily subcutaneous injections of 10–50 nmol of hPTH-(1-30)NH₂/100 g of body weight into young rats starting 2 weeks after OVX significantly increased the femoral trabecular volume and mean thickness of individual trabeculae above those in sham-operated control rats. In OVX rats treated with 50 nmol of hPTH-(1-30)NH₂/100 g of body weight, the trabecular volume was 2.6 times higher and the mean trabecular thickness nearly 4 times higher than in the sham-operated control rats. This very large increase in the mean trabecular thickness was as much as the increase induced by 2 nmol/100 g of body weight of hPTH-(1-31)NH₂, [Leu²⁷]cyclo(Glu²²-Lys²⁶)-hPTH-(1-31)NH₂, hPTH-(1-34)NH₂ and [Leu²⁷]cyclo(Glu²²-Lys²⁶)-hPTH-(1-34)NH₂. These results have removed a major objection to the proposal that PTH's osteogenic action in rats can be triggered solely by intermittent surges of cAMP and the bursts of cAMP-dependent protein kinase activity they cause. Received: 16 September 1998 / Accepted: 15 December 1998     

Prostaglandin E2 (PGE2) Induces the c-fos and c-jun Expressions via the EP1 Subtype of PGE Receptor in Mouse Osteoblastic MC3T3-E1 Cells

This study examined which subtype(s) of PGE receptors is involved in the induction of c-fos and c-jun by PGE₂ in MC3T3-E1 cells. We also investigated the possibility that the induction of these genes is involved in the growth and differentiation of this cell line. PGE₂ dose-dependently induced c-fos and c-jun mRNA expressions in MC3T3-E1 cells. Of the PGE analogs, 17-phenyl-ω-trinor PGE₂ (EP₁ agonist) and sulprostone (EP₁/EP₃ agonist) were far more potent than butaprost (EP₂ agonist) and 11-deoxy PGE₁ (EP₂/EP₄ agonist) in inducing c-fos and c-jun mRNA expressions. Since MC3T3-E1 cells do not express the EP₃ subtype, these results suggest that PGE₂ induces c-fos and c-jun mRNA expressions through the EP₁ subtype of its receptor. In order to study the functional relevance of these protooncogenes, we then studied the effect of inhibition of their synthesis by the use of antisense oligonucleotide. Alkaline phosphatase (ALP) suppression by 17-phenyl-ω-trinor PGE₂ was reversed by antisense oligonucleotide for either c-fos or c-jun. These results suggest that PGE₂, via the EP₁ subtype of the PGE receptor, negatively modulates the transition from proliferation to the matrix maturation stage through the induction of c-fos and c-jun. However, antisense oligonucleotide for c-fos or c-jun did not alter the prostaglandin G/H synthase-2 mRNA expression induced by EP₁. Thus, it is possible that c-fos and c-jun inductions do not account for all the EP₁-mediated PGE₂ actions in MC3T3-E1 cells. Received: 7 October 1998 / Accepted: 30 September 1999