Transcriptional regulation of alpha 2(I) collagen gene expression by fibroblast growth factor-2 in MC3T3-E1 osteoblast-like cells.

Fibroblast growth factor-2 (FGF-2) stimulates proliferation and inhibits differentiated function of osteoblasts by suppressing synthesis of type I collagen and other proteins. However, little is known regarding the molecular mechanisms regulating the suppressive effects of FGF-2 on type I collagen synthesis in osteoblasts. The zinc finger transcription factor Egr-1 and the basic helix-loop-helix (bHLH) family of proteins have been implicated in the regulation of genes crucial to mesodermal cell growth and differentiation. The aim of this study was to determine whether Egr-1 and TWIST might be potential transcriptional regulators of the inhibitory effects of FGF-2 on alpha2(I) collagen expression in MC3T3-E1 osteoblasts which undergo a developmental sequence in vitro. Upon treatment of undifferentiated MC3T3-E1 cells with 1 nM FGF-2, Egr-1 mRNA increased with the effect maximal after 30-60 min. TWIST mRNA also increased with the effect maximal at 2 h. We analyzed the transcriptional control of alpha2(I) collagen gene expression by FGF-2 by transient transfection of an alpha2(I) collagen-luciferase construct (pH5) into undifferentiated MC3T3-E1 cells. The activity of the pH5 luciferase promoter decreased in a dose-dependent manner following treatment with.01 and 1 nM FGF-2. We identified putative Egr-1 and TWIST recognition sequences in the proximal region of the promoter for the murine alpha2(I) collagen gene and a putative Egr-1 site in the 5' region of the murine TWIST promoter. In gel mobility shift assays, potential Egr-1 response elements in the 5' region of the murine TWIST and alpha2(I) collagen genes demonstrated specific Egr-1 binding activity with bFGF-treated nuclear extracts obtained from MC3T3-E1 cells. These results indicate that Egr-1 and TWIST are expressed in undifferentiated MC3T3-E1 osteoblast-like cells following treatment with FGF-2 and they may be potential transcriptional regulators of FGF-2s negative effects on alpha2(I) collagen gene expression. J. Cell. Biochem. 80:550-559, 2001. Published 2001 Wiley-Liss, Inc.

Geropharmacology for the rheumatologist.

Drug administration to older patients with rheumatic disease is a challenge because they are more likely to have adverse drug reactions compared with younger patients. Elderly patients are at risk for adverse drug effects because they often have multiple acute and chronic illnesses and are on several prescription and over-the-counter medications. This article will enhance the practitioner's understanding of how disease and age modulate the pharmacokinetics and pharmacodynamics of medications commonly prescribed for rheumatic disorders. Minimizing the number of drugs prescribed, starting medications at low doses and increasing slowly, and monitoring for toxicity are especially important in the elderly. With judicious use of medications, the quality of life of older patients with rheumatic disease can be enhanced.

Surface adhesion-mediated regulation of chondrocyte-specific gene expression in the nontransformed RCJ 3.1C5.18 rat chondrocyte cell line.

Recent evidence suggests that decreased chondrocyte function in osteoarthritis and other articular disorders may be due to chondrocyte dedifferentiation produced by altered regulatory signals from the cartilage extracellular matrix (ECM). However, there are currently no mammalian chondrocytic cell line systems adapted to the study of this process. We therefore examined the effects of ECM growth conditions on markers of differentiated chondrocytic phenotype expression in the nontransformed rat RCJ 3.1C5.18 (RCJ) chondrocyte cell line, including type II collagen expression, aggrecan production, link protein gene expression, and parathyroid hormone (PTH) receptor number. RCJ cells grown in monolayer on plastic exhibited a dedifferentiated phenotype characterized by flattened cell morphology, with > 80% type I collagen and < 5% type II collagen production, as determined by two-dimensional gel mapping electrophoresis of collagen cyanogen bromide peptides. In addition, aggrecan production was low, and link protein mRNA was not expressed at detectable levels. After transfer to growth under minimal attachment conditions on the surface of a composite type I collagen/agarose (0.15%-0.8%) gel (CAG) for 7 days, RCJ cells developed a rounded, chondrocytic morphology and a pattern of differentiated, chondrocytic gene expression, with 79% type II and 8% type I collagen production. Steady-state type I and type II procollagen mRNA levels were altered in parallel with collagen protein expression. In cells grown on CAG, aggrecan production increased 6-fold, and there was a marked increase in both aggrecan core protein and link protein mRNA levels. In addition, maximal PTH-stimulated cAMP generation increased 15-fold in association with an increased PTH receptor number. Therefore, the RCJ chondrocyte cell line is highly sensitive to ECM regulation of chondrocyte-specific gene expression.

Prostaglandin E2 induces Egr-1 mRNA in MC3T3-E1 osteoblastic cells by a protein kinase C-dependent pathway.

Prostaglandin E2 (PGE2) plays an important role in the regulation of osteoblast metabolism. However, the nuclear signal transduction mechanisms involved in the actions of PGE2 have not been clearly defined. One mechanism may involve induction of immediate early genes such as the transcription factor Egr-1. In the present study, we examined the effects of PGE2 on induction of Egr-1 mRNA in MC3T3-E1 osteoblasts. Time course studies with 2 microM PGE2 showed maximal induction of Egr-1 mRNA at 30 min. In cells pretreated with cycloheximide (CHX), induction of Egr-1 mRNA reached a maximum at 60 min and remained elevated for at least 240 min. Preincubation with CHX was associated with superinduction of Egr-1. Inhibition of protein kinase C activity by pretreatment with 1 microM chelerythrine chloride or by prolonged stimulation with 50 ng/ml tetradecanoyl phorbol acetate (TPA) attenuated the induction of Egr-1 mRNA by 2 microM PGE2. These data indicate that in MC3T3-E1 cells, PGE2 increase Egr-1 mRNA levels via a protein kinase C-dependent pathway.

Epidermal growth factor induces Egr-1 messenger RNA and protein in mouse osteoblastic cells.

The nuclear signaling events activated when epidermal growth factor (EGF) interacts with osteoblasts to produce effects on growth and differentiation are not clearly understood, and may include induction of immediate early genes such as Egr-1, a zinc finger transcription factor. In the present study, Northern analyses were performed to define the effects of EGF on the expression of Egr-1 mRNA in MC3T3-E1 mouse osteoblastic cells. Following treatment of quiescent, subconfluent MC3T3-E1 cells with 0.1-100 ng/ml EGF for various periods, maximal induction of Egr-1 mRNA occurred when cells were treated for 30-60 minutes with 1-10 ng/ml EGF. Inhibition of protein kinase C activity by pretreatment with 1 microM chelerythrine chloride or by prolonged stimulation with 50 ng/ml tetradecanoyl phorbol acetate (TPA) partially diminished the induction of Egr-1 by EGF. Using an immunohistochemical approach, 10 ng/ml EGF was observed to induce Egr-1 protein within 30-60 minutes and this induction was localized to the nucleus. These observations indicate that EGF induces Egr-1 mRNA and protein via protein kinase C and other signaling pathways, and that Egr-1 may be part of the regulatory network mediating the actions of EGF on growth and differentiation of osteoblasts.

The effects of prostaglandin E2, parathyroid hormone, and epidermal growth factor on mitogenesis, signaling, and primary response genes in UMR 106-01 osteoblast-like cells.

Prostaglandin E2 (PGE2), PTH, and epidermal growth factor (EGF) are potent regulators of osteoblast proliferation. In UMR 106-01 rat osteosarcoma cells with osteoblast-like features, PGE2 and PTH inhibit, while EGF stimulates, mitogenesis. Both PGE2 and PTH increase intracellular cAMP levels, cytosolic calcium, and inositol phosphate turnover. In a variety of cell types, EGF mediates its effects in part via activation of receptor protein-tyrosine kinase and other protein kinases, such as protein kinase-C. The nuclear mechanisms of PGE2, PTH, and EGF regulation of osteoblast proliferation are unknown. Accordingly, we have examined the effects of these agents on mitogenesis, second messenger generation, and primary response genes, which may link second messenger activation to subsequent alterations in gene expression. Northern blot analysis of mRNA from UMR 106-01 cells treated for 3 h with 2 microM PGE2, 10 nM PTH, or 10 ng/ml EGF in the presence of cycloheximide demonstrated that all three agents induced the expression of c-fos and c-jun mRNA. In contrast, only EGF stimulated cellular proliferation and induced Egr-1 mRNA. Also, unlike PGE2 and PTH, EGF did not increase intracellular cAMP levels. c-fos mRNA was induced by treatment with 50 ng/ml tetradecanoyl phorbol acetate or by 40 ng/ml forskolin, while induction of Egr-1 mRNA was stimulated by treatment with tetradecanoyl phorbol acetate, but not forskolin. Thus, EGF signal transduction differs from that of PGE2 and PTH in UMR 106-01 osteoblast-like cells, in that EGF does not stimulate the protein kinase-A second messenger system, but causes activation of Egr-1, a primary response gene that may play a role in the mitogenic effect of EGF.

Effects of interleukin-6 on cellular function in UMR-106-01 osteoblastlike cells.

High levels of interleukin-6 (IL-6) have been detected in synovial fluid from patients with inflammatory arthropathies associated with local bone resorption, suggesting a role for IL-6 as a local regulator of bone resorption and remodeling. In the present study we examined the effects of IL-6 on [3H]thymidine ([3H]TdR) incorporation, collagen synthesis, and alkaline phosphatase activity in UMR-106-01 rat osteoblastic osteosarcoma cells. IL-6 stimulated a dose-dependent increase in [3H]TdR incorporation that was maximal at 1000 U/ml (-147% of basal, p less than 0.005) in osteoblastlike cells that were in a logarithmic phase of growth. The increase in [3H]TdR incorporation was maximal between 12 and 24 h and was neutralized by pretreatment with the polyclonal rabbit antibody to IL-6. IL-6 also increased cell number and the secretion of prostaglandin E2 in UMR-106-01 cells in logarithmic growth phase. The stimulation of [3H]TdR incorporation and release of PGE2 into the culture medium by IL-6 was inhibited by indomethacin. A 24 h exposure of the osteoblastlike cells to 1000 U/ml of IL-6 reduced [3H]proline incorporation into collagenase-digestible (CDP) protein to 73% of control values (p less than 0.01). Noncollagen protein (NCP) synthesis was inhibited to 80% of control values (p less than 0.01) by 1000 U/ml of IL-6. The inhibitory effect was relatively greater on CDP than on NCP and consequently resulted in a decrease in the percentage of collagen synthesis. Alkaline phosphatase activity was not altered in these cells after a 24 h exposure to 1-1000 U/ml of IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of nicotine on cellular function in UMR 106-01 osteoblast-like cells.

We examined the effect of nicotine on cellular proliferation, as measured by [3H]thymidine (TdR) incorporation and cell count, and on alkaline phosphatase activity in UMR 106-01 rat osteoblastic osteosarcoma cells. The cells were cultured with varying concentrations of nicotine in serum-free medium for 2 to 72 hours. Nicotine produced a dose-dependent suppression of TdR incorporation, with maximum suppression seen at 10 mM (7% of control); the EC50 for suppression of TdR incorporation was 10 microM. 1 microM nicotine decreased cell number by 20% to 30%. The time course of the effect of 100 microM nicotine on DNA synthesis was measured by TdR incorporation. TdR uptake was measured at 2, 4, 6, 24, 48, and 72 hours. After the addition of nicotine, the following biphasic response in TdR incorporation was observed: a 15% decrease at 2 hours, recovery to near control value at 6 hours, a 27% decrease by 24 hours, and a maximum decrease of 88% by 48 hours. Over a dose range of 1 nM to 10 mM, nicotine produced a dose-dependent increase in alkaline phosphatase activity with maximum stimulation seen at 1 microM (189% of control). We conclude that nicotine suppresses cellular proliferation and stimulates alkaline phosphatase activity in UMR 106-01 osteoblast-like cells. These results may be of significance in the development of osteoporosis and alveolar bone loss associated with the use of tobacco.

Subacute perimyositis.

We describe a patient who developed a benign myopathy with features suggestive of subacute perimysial inflammation. There was no blood eosinophilia or infectious agent identified. Creatine phosphokinase was not significantly elevated. Electromyography showed a myopathic process. Muscle biopsy showed predominantly subacute perimysial inflammation with rare eosinophils. The patient recovered completely after therapy with glucocorticoids and a nonsteroidal antiinflammatory drug. The features of our case were compared to known causes of myositis with or without eosinophils. It may be part of the spectrum of relapsing eosinophilic perimyositis or may be a condition previously unreported.

Polyarteritis nodosa of the epididymis in a patient with Whipple's disease.

The case of a 55-year-old white male who developed necrotizing arteritis localized to the superior pole of the epididymis is presented. He had a history of Whipple's disease and euthyroid Graves' disease. Histopathologic section of an extratesticular mass showed a necrotizing vasculitis with giant cells; periodic acid Schiff stain for Whipple "bacilli" was negative. The combination of rare diseases in our patient suggests the possibility of a common infectious or immune etiology, perhaps mediated via circulating immune complexes.

Technetium-99m MDP demonstration of heterotopic ossification in fibrodysplasia ossificans progressiva.

The extensive heterotopic bone formation in patients with fibrodysplasia ossificans progressiva (FOP) has been documented previously with radiographs. A case in which a Tc-99m MDP bone scan showed increased uptake at sites well before ossification could be documented radiographically is described. This finding suggests that bone scans would likely be useful to monitor the extent of involvement with FOP and to detect areas of new activity.