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Transcriptional activation of the human inducible nitric oxide synthase promoter in osteoblasts
570
Abstracts
Bone Vol. 17, No. 6 December 1995:557-596
48
5O
INTERLEUKIN 11 (IL 11) AND TRANSFORMING GROWTH FACTOR-BETA (TGF-I3) BUT NOT INTERLEUKIN 4 (IL 4) INHIBIT INTERLEUKIN 6 (IL 6) PRODUCTION BY IMMORTALIZED HUMAN BONE MARROW STROMAL CELLS. B.O. Ovaiobi, A. Houc, hton. P. Liu, H. Hasan, B.M.|. Stringer and R.G.G. Russell. Dept of H u n ~ n Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Sheffield $10 2RX, U.K. Previous studies have shown that IL 6, a multifunctional cytokine, is secreted by bone marrow-derived stromal cells and may affect osteoclast formation. IL 11 is another pleiotrophic cytokine which was originally identified and cloned from bone marrow stromal cells and there is increasing evidence that it may also have effects on cells of the osteogenic lineage in addition to its well-documented role in osteoclast development. TGF-13 is also produced by stromal fibroblasts and has been shown to regulate the growth and differentiation of osteoblastic cells. We recently reported the establishment of temperature-sensitive immortalized human bone marrow-derived stromal cell lines [Houghton et al., J. Bone Miner Res 10 (suppl. 1), $313, 1995], one of which (clone 7) expresses markers of the osteoblast phenotype. In the present study, we examined whether IL 11 a n d / o r TGF-~ modulate the secretion of IL 6 by clone 7 cells. Cells seeded at 1.2x 104 cells/cm2 were cultured in txMEM supplemented with 1% FCS and ascorbate at 39°C, in the presence of recombinant human (rh) IL 11 or TGF-[~I alone and in combination with rhILl~ (10U/ml). IL 6 in media conditioned by cells for 24 hours was assayed using a specific ELISA. In comparison to short-term cultures of human bone marrow stromal cells, clone 7 cells constitutively secrete IL 6 at very high levels (> 400U/ml c.f. 25-50U/ml) and this was markely increased in the presence of I L I (> 10 fold). Both TGF-~ (l-50ng/ml) and IL 11 (l-100ng/ml) significantly inhibited constitutive IL 6 production (->30% inhibition) as well IL linduced IL 6 secretion (->50% inhibition). As IL 4, a T-cell product, has been shown to regulate IL 6 production in other cell types such as monocytes, we also examined the effect of this cytokine on IL 6 production by clone 7 cells. In contrast to IL 11 and TGF-[~, rhlL 4 (0.150ng/ml) did not have any effect on either basal or IL 1-induced IL 6 production by these cells. In conclusion, the data provide further evidence that osteogenic cells are targets for IL 11. Furthermore, they suggest that IL 6 production by bone marrow stromal cells is regulated, in part, via an autocrine mechanism. The significance of this remains to be established.
TRANSCRIPTIONAL ACTIVATION OF THE HUMAN INDUCIBLE NITRIC OXIDE SYNTHASE PROMOTER IN OSTEOBLASTS. CJ Petrie. SH Ralston. Department of Medicine and Therapeutics, Foresterhill, Aberdeen AB9 2ZD, UK. Pro-inflammatory cytokines induce nitric oxide production in bone cells and NO has potent effects on osteoblast and osteoclast activity. Our previous work has shown that cytokines stimulate inducible nitric oxide synthase (iNOS) mRNA in human primary osteoblast cultures and in murine MC3T3 osteoblast-like cells. Surprisingly, we failed to detect cytokine induced NO production in several human osteoblast like cell lines, suggesting that transcription may be silenced or that the signalling pathways necessary for iNOS activation may be lacking. In order to investigate this further, we studied transcriptional regulation of the human iNOS promoter by cytokines and glucocorticoids in MG63 human osteosarcoma cells. The iNOS promoter, from -650bp to +50 bp (where +1 is the transcriptional start site) was generated by polymerase chain reaction (PCR) and cloned into a promoterless luciferase reporter vector (PGL2; Promega). The construct was transfected into cultured osteoblasts using liposome mediated DNA transfer, along with PSVI3-Gal, to control for transfection efficiency. Transcriptional activity was assessed by measurement of luciferase activity (corrected for [3-galactosidase) in cell extracts. The iNOS/PGL2 construct stimulated transcription of the reporter gene more than 200-fold compared with the vector alone in MC3T3 cells, even in the absence of cytokine stimulation. Interestingly, reporter activity was not further increased by cytokines, but was significantly supressed by dexamethasone (10 .6 M), a factor known to supress cytokine induced NO production in vitro. In MG63 cells, very low levels of luciferase activity were observed; less than 2% of those achieved when the same construct was transfected into MC3T3 cells. Cytokine stimulation did not increase luciferase activity in the MG63 cells. These findings suggest that factors which are constitively expressed in MC3T3 cells may cause activation of the iNOS promoter, and that the elements necessary for repression of iNOS transcription by dexamethasone are present within the first 600bp. The low levels of transcription in MG63 cells even after cytokine stimulation is consistent with previous findings, and suggest that MG63 cells may not possess the signalling pathways or transcriptional machinery to cause iNOS activation.
49
50a
INFLUENCE OF RETINOIDS ON PROLIFERATION OF PRIMARY HUMAN OSTEOBLASTS IN VITRO
OSTEOBLASTIC DIFFERENTIATION REDUCES SERUM INDUCED TRANSCRIPTION OF PROSTAGLANDIN G/H SYNTHASE-2 IN MC3T3-E1 CELLS C.C. Plbeam, O.S. Voznesensky, C.B. Alander and L.G. Raisz. University of Connecticut Health Center, Farmington CT 06030 USA. Prostaglandin G/H synthase-2 (PGHS-2), the major enzyme in the conversion of arachidonic acid to prostaglandins (PG), is an early response gene which mediates the acute regulation of PGs by cytokines, growth factors and hormones. To examine the effects of differentiation on PGHS-2 expression, we used MC3T3-E1 cells stably transfected with -371/+70 bp of the PGHS-2 promoter fused to a luciferase reporter. These constructs were the generous gift of Dr. H. Herschman (UCLA). Cells were plated at 5000/cm 2 in DMEM with 10% fetal calf serum (FCS) and cultured up to 4 weeks; phosphoascorbate was added affer the first week to facilitate collagen synthesis, mRNA levels were measured by Northern analysis. Luciferase activity was measured in soluble cell extracts by luminometer and normalized to total protein. Medium PGE2 was measured by RIA. Cells were serum-deprived for 24 h before 2-3 h of treatment with agonists, mRNA levels for osteocalcin and type I collagen, markers for osteoblastic differentiation, were increased after two weeks of culture. PGHS-2 mRNA was not expressed in serumdeprived cells but was induced by 2 h of serum (FCS, 10%), forskolin (FSK, 10.5 M), phorbol myristate acetate (PMA, 10-e M), and PGE2 (10~ M). At 1 wk of culture, serum was the most potent inducer of PGHS-2 mRNA. After 2 weeks of culture, the induction by serum was markedly reduced, with little change in the following weeks. Similarly, the 2 h serum stimulation of PGE2 production was decreased by 7-10 fold after 2 wk. In contrast, the induction of PGHS-2 mRNA by FSK, PMA, or PGE2 was maintained, or increased, after 2-4 weeks of culture. Changes in luciferase activity, measured 3 h after treatment with agonists, paralleled the mRNA changes, indicating that the reduction of the serum induction of PGHS-2 expression with increasing differentiation was transcriptionally mediated. These results indicate that less differentiated cells in the early replicative phase respond most strongly to the growth factors in serum. We speculate that high levels of endogenous PGs may play a role in initiating differentiation in osteoblastic cells.
L.Pecur, K.Weber. G.Leb. R.Wildburaer and G.J.Kreis. Department of Medicine, KarI-Franzens University of Graz, Auenbruggerplatz 15, A-8036 Graz Introduction: Retinoids are used in treatment of rheumatologic and skin diseases as well as in prevention and treatment of cancer. However, side-effects such as spinal hyperostoses (bone spurs), disorders similar to diffuse idiopathic skeletal hyperostosis and tendon and ligament calcification have been observed in patients receiving long-term therapy. We investigated the influence of four retinoids on proliferation of human osteoblasts. M e t h o d s : Primary cell culture was established from female trabecular bone using mechanical and enzymatic technique. AII-trans retinoic acid, 9-cis retinoic acid, 13-cis retinoic acid and acitretin (lpM-lmM) were added to cultured osteoblasts in 96-well plates for 48 hours. Growth stimulation was measured by (3H)thymidine incorporation. Results:13-cis RA stimulated osteoblast proliferation within a wide range of 10pM-lmM. Both, all-trans RA and 9-cis RA enhanced proliferation ratio (p<0.05) within range of 10nMl mM, whereas acitretin inhibited osteoblast proliferation in raising concentrations fromlnM to l m M . Conclusion: 13-cis retinoic acid (isotretinoin) therapy is associated with developing spinal and extraspinal hyperostoses. By constant stimulation, and even in wide range of therapeutic concentrations, human osteoblasts might be enhanced to proliferate thus forming bone protrusions.
Abstracts
Bone Vol. 17, No. 6 December 1995:557-596
48
5O
INTERLEUKIN 11 (IL 11) AND TRANSFORMING GROWTH FACTOR-BETA (TGF-I3) BUT NOT INTERLEUKIN 4 (IL 4) INHIBIT INTERLEUKIN 6 (IL 6) PRODUCTION BY IMMORTALIZED HUMAN BONE MARROW STROMAL CELLS. B.O. Ovaiobi, A. Houc, hton. P. Liu, H. Hasan, B.M.|. Stringer and R.G.G. Russell. Dept of H u n ~ n Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Sheffield $10 2RX, U.K. Previous studies have shown that IL 6, a multifunctional cytokine, is secreted by bone marrow-derived stromal cells and may affect osteoclast formation. IL 11 is another pleiotrophic cytokine which was originally identified and cloned from bone marrow stromal cells and there is increasing evidence that it may also have effects on cells of the osteogenic lineage in addition to its well-documented role in osteoclast development. TGF-13 is also produced by stromal fibroblasts and has been shown to regulate the growth and differentiation of osteoblastic cells. We recently reported the establishment of temperature-sensitive immortalized human bone marrow-derived stromal cell lines [Houghton et al., J. Bone Miner Res 10 (suppl. 1), $313, 1995], one of which (clone 7) expresses markers of the osteoblast phenotype. In the present study, we examined whether IL 11 a n d / o r TGF-~ modulate the secretion of IL 6 by clone 7 cells. Cells seeded at 1.2x 104 cells/cm2 were cultured in txMEM supplemented with 1% FCS and ascorbate at 39°C, in the presence of recombinant human (rh) IL 11 or TGF-[~I alone and in combination with rhILl~ (10U/ml). IL 6 in media conditioned by cells for 24 hours was assayed using a specific ELISA. In comparison to short-term cultures of human bone marrow stromal cells, clone 7 cells constitutively secrete IL 6 at very high levels (> 400U/ml c.f. 25-50U/ml) and this was markely increased in the presence of I L I (> 10 fold). Both TGF-~ (l-50ng/ml) and IL 11 (l-100ng/ml) significantly inhibited constitutive IL 6 production (->30% inhibition) as well IL linduced IL 6 secretion (->50% inhibition). As IL 4, a T-cell product, has been shown to regulate IL 6 production in other cell types such as monocytes, we also examined the effect of this cytokine on IL 6 production by clone 7 cells. In contrast to IL 11 and TGF-[~, rhlL 4 (0.150ng/ml) did not have any effect on either basal or IL 1-induced IL 6 production by these cells. In conclusion, the data provide further evidence that osteogenic cells are targets for IL 11. Furthermore, they suggest that IL 6 production by bone marrow stromal cells is regulated, in part, via an autocrine mechanism. The significance of this remains to be established.
TRANSCRIPTIONAL ACTIVATION OF THE HUMAN INDUCIBLE NITRIC OXIDE SYNTHASE PROMOTER IN OSTEOBLASTS. CJ Petrie. SH Ralston. Department of Medicine and Therapeutics, Foresterhill, Aberdeen AB9 2ZD, UK. Pro-inflammatory cytokines induce nitric oxide production in bone cells and NO has potent effects on osteoblast and osteoclast activity. Our previous work has shown that cytokines stimulate inducible nitric oxide synthase (iNOS) mRNA in human primary osteoblast cultures and in murine MC3T3 osteoblast-like cells. Surprisingly, we failed to detect cytokine induced NO production in several human osteoblast like cell lines, suggesting that transcription may be silenced or that the signalling pathways necessary for iNOS activation may be lacking. In order to investigate this further, we studied transcriptional regulation of the human iNOS promoter by cytokines and glucocorticoids in MG63 human osteosarcoma cells. The iNOS promoter, from -650bp to +50 bp (where +1 is the transcriptional start site) was generated by polymerase chain reaction (PCR) and cloned into a promoterless luciferase reporter vector (PGL2; Promega). The construct was transfected into cultured osteoblasts using liposome mediated DNA transfer, along with PSVI3-Gal, to control for transfection efficiency. Transcriptional activity was assessed by measurement of luciferase activity (corrected for [3-galactosidase) in cell extracts. The iNOS/PGL2 construct stimulated transcription of the reporter gene more than 200-fold compared with the vector alone in MC3T3 cells, even in the absence of cytokine stimulation. Interestingly, reporter activity was not further increased by cytokines, but was significantly supressed by dexamethasone (10 .6 M), a factor known to supress cytokine induced NO production in vitro. In MG63 cells, very low levels of luciferase activity were observed; less than 2% of those achieved when the same construct was transfected into MC3T3 cells. Cytokine stimulation did not increase luciferase activity in the MG63 cells. These findings suggest that factors which are constitively expressed in MC3T3 cells may cause activation of the iNOS promoter, and that the elements necessary for repression of iNOS transcription by dexamethasone are present within the first 600bp. The low levels of transcription in MG63 cells even after cytokine stimulation is consistent with previous findings, and suggest that MG63 cells may not possess the signalling pathways or transcriptional machinery to cause iNOS activation.
49
50a
INFLUENCE OF RETINOIDS ON PROLIFERATION OF PRIMARY HUMAN OSTEOBLASTS IN VITRO
OSTEOBLASTIC DIFFERENTIATION REDUCES SERUM INDUCED TRANSCRIPTION OF PROSTAGLANDIN G/H SYNTHASE-2 IN MC3T3-E1 CELLS C.C. Plbeam, O.S. Voznesensky, C.B. Alander and L.G. Raisz. University of Connecticut Health Center, Farmington CT 06030 USA. Prostaglandin G/H synthase-2 (PGHS-2), the major enzyme in the conversion of arachidonic acid to prostaglandins (PG), is an early response gene which mediates the acute regulation of PGs by cytokines, growth factors and hormones. To examine the effects of differentiation on PGHS-2 expression, we used MC3T3-E1 cells stably transfected with -371/+70 bp of the PGHS-2 promoter fused to a luciferase reporter. These constructs were the generous gift of Dr. H. Herschman (UCLA). Cells were plated at 5000/cm 2 in DMEM with 10% fetal calf serum (FCS) and cultured up to 4 weeks; phosphoascorbate was added affer the first week to facilitate collagen synthesis, mRNA levels were measured by Northern analysis. Luciferase activity was measured in soluble cell extracts by luminometer and normalized to total protein. Medium PGE2 was measured by RIA. Cells were serum-deprived for 24 h before 2-3 h of treatment with agonists, mRNA levels for osteocalcin and type I collagen, markers for osteoblastic differentiation, were increased after two weeks of culture. PGHS-2 mRNA was not expressed in serumdeprived cells but was induced by 2 h of serum (FCS, 10%), forskolin (FSK, 10.5 M), phorbol myristate acetate (PMA, 10-e M), and PGE2 (10~ M). At 1 wk of culture, serum was the most potent inducer of PGHS-2 mRNA. After 2 weeks of culture, the induction by serum was markedly reduced, with little change in the following weeks. Similarly, the 2 h serum stimulation of PGE2 production was decreased by 7-10 fold after 2 wk. In contrast, the induction of PGHS-2 mRNA by FSK, PMA, or PGE2 was maintained, or increased, after 2-4 weeks of culture. Changes in luciferase activity, measured 3 h after treatment with agonists, paralleled the mRNA changes, indicating that the reduction of the serum induction of PGHS-2 expression with increasing differentiation was transcriptionally mediated. These results indicate that less differentiated cells in the early replicative phase respond most strongly to the growth factors in serum. We speculate that high levels of endogenous PGs may play a role in initiating differentiation in osteoblastic cells.
L.Pecur, K.Weber. G.Leb. R.Wildburaer and G.J.Kreis. Department of Medicine, KarI-Franzens University of Graz, Auenbruggerplatz 15, A-8036 Graz Introduction: Retinoids are used in treatment of rheumatologic and skin diseases as well as in prevention and treatment of cancer. However, side-effects such as spinal hyperostoses (bone spurs), disorders similar to diffuse idiopathic skeletal hyperostosis and tendon and ligament calcification have been observed in patients receiving long-term therapy. We investigated the influence of four retinoids on proliferation of human osteoblasts. M e t h o d s : Primary cell culture was established from female trabecular bone using mechanical and enzymatic technique. AII-trans retinoic acid, 9-cis retinoic acid, 13-cis retinoic acid and acitretin (lpM-lmM) were added to cultured osteoblasts in 96-well plates for 48 hours. Growth stimulation was measured by (3H)thymidine incorporation. Results:13-cis RA stimulated osteoblast proliferation within a wide range of 10pM-lmM. Both, all-trans RA and 9-cis RA enhanced proliferation ratio (p<0.05) within range of 10nMl mM, whereas acitretin inhibited osteoblast proliferation in raising concentrations fromlnM to l m M . Conclusion: 13-cis retinoic acid (isotretinoin) therapy is associated with developing spinal and extraspinal hyperostoses. By constant stimulation, and even in wide range of therapeutic concentrations, human osteoblasts might be enhanced to proliferate thus forming bone protrusions.