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(Circulation Research. 1999;85:1124.)
© 1999 American Heart Association, Inc.

Cellular Biology

Oncostatin M Induces Interleukin-6 and Cyclooxygenase-2 Expression in Human Vascular Smooth Muscle Cells

Synergy With Interleukin-1ß

Catherine Bernard, Régine Merval, Marilyne Lebret, Philippe Delerive, Isabelle Dusanter-Fourt, Stéphanie Lehoux, Christophe Créminon, Bart Staels, Jacques Maclouf¹, Alain Tedgui

From the Institut National de la Santé et de la Recherche Médicale U141 (C.B., R.M., S.L., A.T.), Institut National de la Santé et de la Recherche Médicale U348 (M.L., J.M.), and Institut Fédératif de Recherche Circulation, Hôpital Lariboisière, Paris; Institut National de la Santé et de la Recherche Médicale U325 (P.D., B.S.), Institut Pasteur, Lille; Institut National de la Santé et de la Recherche Médicale U367, Hôpital Cochin (I.D.-F.), Paris; and Commissariat à l’Energie Atomique (C.C.), Gif-sur-Yvette, France.

Correspondence to Dr Alain Tedgui, INSERM U141, 41 Blvd de la Chapelle, 75475 Paris Cedex 10, France. E-mail tedgui{at}infobiogen.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—Oncostatin M (OSM), a cytokine first identified from activated monocytes and T lymphocytes, is one of the most potent autocrine growth factor for AIDS and Kaposi’s sarcoma. Little is known about the effects of OSM on normal vascular cells. We thus exposed human aortic smooth muscle cells (hASMCs) to OSM, examined cell proliferation and morphology, and determined interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) expression. OSM had a weak antiproliferative effect. After a 4-day incubation with 100 ng/mL OSM, cell count decreased to 69±3% of control. However, OSM induced striking changes in hASMC morphology, characterized by a polyclonal shape, in contrast to the spindle morphological feature of control hASMCs. OSM stimulated the release of IL-6 by hASMCs in a dose-dependent way; after a 48-hour exposure, values were 8.5±0.7, 29.7±3.5, 50.9±4.4, and 73.8±7.6x10³ U/mL (n=6) at OSM concentrations of 0, 1, 10, and 100 ng/mL, respectively. OSM induced marked expression of COX-2 protein and mRNA. Leukemia inhibitory factor had no effect on hASMCs, indicating that OSM effects on hASMCs were mediated by the OSM type II receptor and not by the leukemia inhibitory factor receptor. OSM used the JAK/STAT signaling pathway, as demonstrated by rapid phosphorylation of JAK1 and specific activation of STAT1. Interestingly, OSM acted in synergy with IL-1ß on IL-6 production and COX-2 expression. In conclusion, OSM is a novel regulator of human smooth muscle cell functions, acting in concert with IL-1ß, and OSM may play a role in major vascular diseases such as atherosclerosis.

Key Words: cells, smooth muscle • oncostatin M • JAK/STAT • growth factors • cytokine

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytokine networks are involved in the pathogenesis and progression of numerous vascular diseases, such as atherosclerosis, immune arteritis, and Kaposi’s sarcoma (KS). Oncostatin M (OSM) is a member of interleukin (IL)-6–related cytokines that exhibit a similar helical structure and share receptor components.¹ OSM was first identified from activated human T cells and monocytes.² Subsequently, OSM was characterized as a growth regulator for different cell types, being able to either inhibit or stimulate the cell proliferation.³ OSM is one of the most potent autocrine and paracrine growth factors for AIDS-KS cells.^{4 5 6} KS is a vascular neoplasm, in which proliferative spindle cells are believed to be of endothelial origin. These KS cells are surrounded by immunoinflammatory cells and prominent neovascular elements.⁷ The effect of OSM on the growth of KS cells has been shown to be potentiated by basic fibroblast growth factor, tumor necrosis factor-ß, and IL-6, as well as by the HIV-1 Tat protein.^{6 8}

The potential effects of OSM on normal vascular cells have been poorly explored. OSM, like many cytokines, may display pleiotropic biological activities that encompass pathways leading to proliferation. A few studies report that OSM is able to modulate endothelial properties, such as morphological changes leading to spindle-shaped cells⁸ and protein synthetic phenotype, such as plasminogen activator activity, expression of P-selectin, and IL-6 production.^{9 10}

However, little is known about the effects of OSM on smooth muscle cells, which outnumber the endothelial cells in the type of vessels (arteries, arterioles, or venules) involved in major vascular diseases and are known to produce large amounts of prostaglandins and IL-6.^{11 12}

We therefore studied the effects of OSM on human vascular smooth muscle cell activation by measuring IL-6 production and cyclooxygenase-2 (COX-2) expression, especially in the presence of IL-1ß, as a model of cytokine network.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Cultures
Human aortic smooth muscle cells (hASMCs) from Clonetics were obtained from 2 donors. Cells were used between passages 4 and 8. hASMCs were grown in culture medium supplemented with 5% FBS, 5 µg/mL insulin, 2 ng/mL recombinant human basic fibroblast growth factor, 10 ng/mL recombinant human epidermal growth factor, 50 µg/mL gentamycin, and 50 ng/mL amphotericin B. hASMCs were cultured until subconfluence was reached and then incubated in complete culture medium with 5% FBS before exposure for a subsequent period of time in the presence of the different cytokines and reagents that were tested.

Mitogenic Assay and Cell Morphology Analysis
The proliferation of hASMCs was assessed on the basis of cell number. Proliferating or subconfluent cells were made quiescent for 24 hours in serum-free culture medium. After being replenishing with fresh culture medium with or without 5% FBS, cells were further incubated up to 4 days in the absence or presence of OSM (1, 10, or 100 ng/mL) with or without 1 µmol/L NS-398, a specific COX-2 inhibitor.¹³

Focal adhesions were analyzed through the use of vinculin staining. Cells grown onto glass slides at 50% confluence were incubated for 4 days in the absence or presence of OSM (10 ng/mL). They were fixed in 4% paraformaldehyde solution and immunostained with anti-mouse vinculin antibodies (Sigma Chemical Co).

IL-6 Bioassay
IL-6 activity was measured through the use of a specific cell proliferation bioassay with an IL-6–dependent B9 hybridoma cell line,¹⁴ as previously described.¹⁵

RNA Analysis
RNA extractions¹⁶ and Northern blot analysis¹⁷ of total cellular RNA were performed as described; 350 bp of COX-2 cDNA¹⁸ and 240 bp of GAPDH were used as probes.

Western Blot Analysis
hASMCs were washed then lysed in 20 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, 30 mmol/L octyl glucoside, and 0.5 mmol/L Pefabloc for 30 minutes. Equal amounts of protein (30 µg) were subjected to SDS–bisacrylamide gel electrophoresis under reducing conditions.¹⁹ For COX-1 and COX-2 detections, we used monoclonal antibodies specific to either human COX-1 or human COX-2.¹⁹

Immunoprecipitation
hASMCs were incubated in the presence of 10 ng/mL OSM for 0, 10, or 30 minutes at 37°C. Cell extracts were immunoprecipitated overnight in the presence of anti–Janus kinase 1 (JAK1) antibodies and protein A/G agarose (40 µL/mL lysate; both Santa Cruz). Nitrocellulose membranes were incubated in a solution containing phosphotyrosine (PY20) or JAK1 polyclonal antibodies (Santa Cruz). Positive bands were revealed by enhanced chemiluminescence.

Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared as previously described.²⁰ The extracts were incubated for 30 minutes at 4°C with 10 to 15 fmol of a ³²P-labeled oligonucleotide. Complexes were separated on a 4% nondenaturating polyacrylamide gel as reported previously.²⁰ The oligonucleotide probes used (m67 SIE, 5'-CATTTCCCGTAAATC-3'; IRF1-GAS, 5'-GATCCATTTCCCCGAAATGA-3') were end-labeled with the use of T4 polynucleotide kinase. As a positive control for signal transducer and activator of transcription-5 (STAT5) activation, we used nuclear extracts from human hematopoietic UT7-mpl cells treated with thrombopoietin for 10 minutes.²⁰

Prostaglandin E₂ and 6-Keto-Prostaglandin F-1 Assays
Prostaglandin (PG)E₂ and 6-keto-PGF-1 were determined in hASMC supernatants through the use of immunoassay with acetylcholinesterase-labeled PGE₂ and 6-keto-PGF-1 as tracers.¹⁹

Statistical Analysis
Values are presented as mean±SEM. The effects of OSM, IL-1, or both were tested with the use of ANOVA or Student’s t test.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mitogenic and Morphological Effects of OSM
The effect of OSM (1, 10, and 100 ng/mL) on cell proliferation was assessed in proliferative hASMCs in the presence of FBS. After 24 or 48 hours, OSM had no significant effect on cell count at 1 ng/mL (95±3% and 89±6% of control, respectively) or at 10 ng/mL (98±2% and 88±9% of control), but OSM significantly decreased cell count at 100 ng/mL (73±3% and 69±3% of control, P<0.05). After 4 days, the antiproliferative effect of OSM was observed at 10 and 100 ng/mL (79±6% and 74±4% of control, P<0.05) but not at 1 ng/mL (89±5%). NS-398, a specific COX-2 inhibitor, did not influence hASMC counts or cell morphology up to 96 hours. Similar results were observed when FBS was omitted and cells were tested at subconfluence.

In contrast to the slight antiproliferative effect of OSM, striking changes were noted in the hASMC morphology. Although control hASMCs cultured in complete medium with or without FBS were spindle shaped (Figures 1A and 1C), hASMCs cultured in the presence of OSM assumed a polygonal shape (Figures 1B and 1D). IL-1 stimulation did not modify hASMC morphology, but when the cells were incubated in the presence of both OSM and IL-1, they assumed the OSM-induced polygonal shape. The effect of OSM on cell shape was further analyzed through an examination of the changes in focal adhesions. OSM-activated cells were characterized on the basis of the vinculin-rich area of focal contacts (Figure 2).

View larger version (214K): [in this window] [in a new window]   Figure 1. Morphological effect of OSM on hASMCs. Cells were grown until 50% confluence in complete medium supplemented with 5% FBS, made quiescent through incubation in serum-free medium for 24 hours, and then incubated for 4 days with (A and B) or without 5% FBS (C and D) in presence (B and D) or absence of 10 ng/mL OSM (A and C). Cells were observed under phase contrast optics (x40).

View larger version (65K): [in this window] [in a new window]   Figure 2. Vinculin staining of hASMCs incubated for 4 days without (A) or with 10 ng/mL OSM (B) in presence of 5% FBS to identify focal contacts. Abundant vinculin-rich areas of focal contacts were observed in OSM-treated cells (x400).

Effect of OSM on IL-6 Production
OSM stimulated the release of IL-6 by hASMCs in a dose- and time-dependent manner. OSM stimulated IL-6 production with doses as low as 1 ng/mL (Figure 3A). IL-6 activity was stimulated 8 hours after the addition of OSM and continued to increased up to 72 hours (Figure 3B).

View larger version (25K): [in this window] [in a new window]   Figure 3. A, Dose-dependent effect of OSM on IL-6 release by hASMCs incubated for 48 hours. Results are mean of 6 individual experiments. B, Time course of IL-6 release by hASMCs incubated in presence of 10 ng/mL OSM. Results are representative of 3 different experiments.

Effect of OSM on COX-2 Expression
COX-2 expression was induced by increasing doses of OSM (Figure 4A). COX-1 expression was unaffected by OSM. The expression was observed after an 8-hour exposure to OSM, persisted up to 48 hours, and was markedly decreased at 72 hours (Figure 4B).

View larger version (55K): [in this window] [in a new window]   Figure 4. A, Dose-dependent effect of OSM on COX-1 and COX-2 expression in hASMCs. Western analysis of lysates from cells stimulated with OSM for 24 hours. Membranes used for COX-2 data were stripped and COX-1 immunodetection was performed with anti-COX-1 antibodies. Results are representative of 3 different experiments. B, Time-dependent expression of COX-2 in hASMCs. Western analysis of lysates from cells stimulated with 1 ng/mL OSM. Results are representative of 3 different experiments. C, Time course of OSM-induced COX-2 mRNA expression. hASMCs were stimulated with 1 ng/mL OSM for 3, 9, 24, and 48 hours. Northern blot analysis was performed with a COX-2 cDNA probe, as described in Materials and Methods. The blot was stripped and reprobed with a probe for GAPDH for normalization.

Northern blot analysis showed a single hybridization band of 4300 nucleotides (Figure 4C), which is in agreement with the size of COX-2 mRNA previously reported.¹⁸ OSM induced COX-2 mRNA expression after 3 hours, which persisted up to 48 hours (Figure 4C), which is in agreement with the protein expression.

The expression of COX-2 was associated with increased production of prostaglandins: 6-keto-PGF-1 concentrations in cell supernatants after 24-hour incubation with OSM (10 ng/mL) were 141.7±6.8 ng/mL versus 25.8±5.2 ng/mL in control (n=3, P<0.01), and PGE₂ concentrations were 123.7±27.5 ng/mL versus 30.1±3.5 ng/mL (P<0.01), respectively. To assess COX-2 activity, hASMCs were activated with OSM (10 ng) for 24 hours, then washed and reincubated for 30 minutes in the presence of arachidonic acid (10 µmol/L). 6-keto-PGF-1 production was 41.4±6.2 ng/mL versus 13.6±5.2 ng/mL in control (n=3, P<0.05), and PGE₂ production was 47.3±9.1 ng/mL versus 14.9±1.5 ng/mL, respectively (P<0.05).

OSM Activating Pathways
OSM is known to bind with high affinity both OSM receptor (type II) and leukemia inhibitory factor (LIF) receptor (type I), whereas LIF only binds type I receptor. However, LIF had no effect on COX-2 expression by hASMCs (Figure 5A) or IL-6 release (data not shown). Nevertheless, we found that LIF was able to activate KB epidermoid cells (gift of Dr H. Gascan), which express the LIF receptor/gp190²¹ to produce large amounts of IL-6 (data not shown), indicating that the LIF used in our experiments was biologically active. On the other hand, we did not observe any synergy or antagonism between LIF and OSM. Moreover, antibodies neutralizing type II receptor–dependent OSM activity abolished the effects of OSM on hASMCs (Figure 5B). Irrelevant rabbit IgG had no effect, and anti-OSM antibodies had no effect on the production of IL-6 by KB cells. Altogether, these findings suggest that the effect of OSM was mediated by its type II receptor.

View larger version (41K): [in this window] [in a new window]   Figure 5. A, No effect of LIF at doses from 1 to 300 ng/mL on COX-2 expression in hASMCs. Western analysis of lysates from cells stimulated with OSM (10 ng/mL) or LIF for 24 hours. B, Effect of increasing doses of antibodies neutralizing type II receptor–dependent OSM activity (OSM Ab) on COX-2 expression in hASMCs. Western analysis of lysates from cells stimulated with OSM (10 ng/mL) for 24 hours in presence or absence of OSM antibody. C, Inhibitory effect of tyrosine kinase inhibitor genistein on COX-2 expression in hASMCs. Western analysis of lysates from cells stimulated with OSM (10 ng/mL) for 24 hours in presence or absence of genistein at concentration of 10 or 30 µmol/L. hASMCs were preincubated for 1 hour in presence of genistein before stimulation with OSM. Results are representative of 3 different experiments.

The tyrosine kinase inhibitor genistein (at 30 µmol/mL) inhibited IL-6 release induced by OSM by 75.3±7.8% and exerted a marked dose-dependent inhibitory effect on COX-2 expression (Figure 5C), indicating the activation of tyrosine kinase or kinases in the OSM signaling pathway in hASMCs. Daidzein, a structural analog of genistein with no tyrosine kinase inhibitory activity,²² had no effect on IL-6 and COX-2 expression induced by OSM in hASMCs.

We next investigated the signal transduction pathway mediating the secretion of IL-6 and the induction of COX-2 by OSM. Receptors for OSM, related to the IL-6 receptor/gp130 subfamily, belong to the cytokine receptor superfamily. After binding their respective cytokine, these receptors trigger a rapid and strong activation of the JAK/STAT pathway. We therefore tested whether OSM induces the activation of JAK and STAT factors in hASMCs. Figure 6A shows that OSM induced a rapid phosphorylation of JAK1. The presence of activated STAT factors was evaluated with the use of electrophoretic mobility shift assay (EMSA) with 2 different STAT-responsive elements as probes. As shown in Figure 6B, OSM induced a rapid and intense activation of STAT factors (lanes 1 to 4) that bound to either the m67 SIE or the IRF1-GAS probes. To identify the STAT factors that were activated, nuclear extracts were next incubated with the labeled probes in the presence of specific anti-STAT antibodies (supershift assays). As shown in lanes 5 and 6, anti-STAT1, but not anti-STAT3, antibodies were able to recognize and completely shift the nuclear complexes induced by OSM, indicating that the major STAT factor activated by OSM in hASMCs was STAT1. Although OSM and cytokines related to OSM such as IL-6 could induce the activation of STAT5 factors, we observed that STAT complexes activated by OSM in hASMCs did not migrate with the mobility of DNA-bound STAT5 complexes as illustrated in lane 7, showing activated STAT1 and STAT5 in human hematopoietic UT7-mpl cells treated with thrombopoietin for 10 minutes.²⁰

View larger version (41K): [in this window] [in a new window]   Figure 6. A, OSM-induced tyrosine phosphorylation of JAK1. hASMCs were stimulated with 1 ng/mL OSM for indicated times. Immunoprecipitation was performed overnight in presence of anti-JAK1 antibodies, and Western blot analysis was performed with anti-PY20 antibodies. The same blot was probed with anti-JAK1 antibodies to determine equal loading. IP indicates immunoprecipitation. B, Specific activation of STAT1 in hASMCs in response to OSM. Presence of activated STAT factors in nuclear extracts from OSM-activated hASMCs was evaluated with the use of EMSA with 2 different STAT-responsive elements as probes (m67 SIE or IRF1-GAS), as described in Materials and Methods. To identify STAT factors that were activated, nuclear extracts were next incubated with labeled probes in presence of specific anti-STAT antibodies (supershift assays). C indicates control showing activated STAT1 and STAT5 in human hematopoietic UT7-mpl cells as described in Materials and Methods.

To further support the direct involvement of STAT1 in COX-2 and IL-6 expression, hASMCs were incubated in the presence of fludarabine, which has been shown recently to specifically inhibit STAT1 activation.²³ Fludarabine (50 µmol/L) inhibited OSM-induced STAT 1 activation (Figure 7A) and COX-2 expression (Figure 7B). OSM-stimulated IL-6 release was also significantly decreased (by 45±9%) in the presence of fludarabine.

View larger version (47K): [in this window] [in a new window]   Figure 7. A, Effect of fludarabine on STAT1 activation in hASMCs. hASMCs were preincubated for 24 hours in presence of 50 µmol/L fludarabine and then stimulated with 10 ng/mL OSM for 30 minutes. Presence of activated STAT factors in nuclear extracts from hASMCs was evaluated with the use of EMSA with m67 SIE as STAT-responsive element. B, Effect of fludarabine on COX-1 and COX-2 expression in hASMCs. hASMCs were preincubated for 24 hours in presence of 50 µmol/L fludarabine and then stimulated with 10 ng/mL OSM for 24 hours. Membranes used for COX-2 data were stripped, and COX-1 immunodetection was performed with anti-COX-1 antibodies. Results are representative of 2 different experiments. Fludarabine inhibited COX-2 expression but had no effect on COX-1 expression.

Synergy Between OSM and IL-1
OSM acted synergistically with IL-1 on IL-6 release (Figure 8) and COX-2 expression (Figure 9A). The expression of COX-2 by hASMCs stimulated by IL-1 (0.1 ng/mL) was maximum at 8 hours, persisted at 24 hours, and decreased thereafter (Figure 7A). When cells were activated by both OSM (1 ng/mL) and IL-1 (0.1 ng/mL), COX-2 expression was dramatically increased, being already pronounced at 8 hours, and remained expressed at a high level even after 72 hours (Figure 9A). COX-1 expression was not affected by the coincubation with OSM and IL-1. OSM and IL-1 markedly increased COX-2 mRNA level compared with the effect of each cytokine up to 48 hours (Figure 9B), but the effect appeared to be additive and not synergistic. When cells were pretreated for 90 minutes with actinomycin-D (5 µg/mL) to block RNA polymerase II activity and subsequently incubated for 90 minutes with OSM, IL-1, or both, the induction of COX-2 mRNA expression by these compounds was completely abolished, demonstrating that both IL1 and OSM regulate COX-2 expression at the transcriptional level in short-term incubations. When cells were pretreated with OSM, IL-1, or both for 90 minutes and RNA polymerase II activity was subsequently blocked with actinomycin-D, mRNA transcript levels were not significantly affected up to 6 hours thereafter (Figure 10). However, after a 12-hour incubation with actinomycin-D, COX-2 mRNA was almost unchanged in OSM- or OSM-plus-IL-1–treated cells but decreased significantly in IL-1–treated cells (43±5%). The effect of OSM on mRNA stability was further observed after a 24-hour incubation with actinomycin-D: COX-2 mRNA slightly decreased in OSM- or OSM-plus-IL-1–treated cells (9.5±2.5% and 25.5±1.5%, respectively), whereas it markedly decreased in IL-1–treated cells (61±3%).

View larger version (33K): [in this window] [in a new window]   Figure 8. Synergistic effect of IL-1ß (0.1 ng/mL) and OSM (1 ng/mL) on IL-6 release by hASMCs incubated for 48 hours. Results are mean of 3 individual experiments. Ctl indicates control.

View larger version (42K): [in this window] [in a new window]   Figure 9. A, Synergistic effect of IL-1 and OSM on COX-2 expression in hASMCs. Western blot analysis of lysates from cells stimulated with OSM (1 ng/mL), IL-1ß (0.1 ng/mL), or both for 8, 24, and 72 hours. Membranes used for COX-2 data at 72 hours were stripped, and COX-1 immunodetection was performed with anti-COX-1 antibodies. Results are representative of 3 different experiments. B, Increased COX-2 mRNA expression in hASMCs activated by OSM and IL-1. hASMCs were stimulated with 1 ng/mL OSM, 0.1 ng/mL IL-1ß, or both for 3 hours. Northern blot analysis was performed with a COX-2 cDNA probe as described in Materials and Methods. The blot was stripped and reprobed with a probe for GAPDH for normalization.

View larger version (22K): [in this window] [in a new window]   Figure 10. Time course of COX-2 mRNA decay. hASMCs were incubated with OSM (1 ng/mL), IL-1 (0.1 ng/mL), or OSM plus IL-1 for 90 minutes, and then actinomycin-D (5 µg/mL) was added and COX-2 mRNA was determined after 6, 12, and 24 hours. Northern blot analysis was performed with a COX-2 cDNA probe as described in Materials and Methods. The blot was stripped and reprobed with a probe for GAPDH for normalization. The ratio of COX-2 to GAPDH ratio at time 0 was taken as 100%. Results are mean of 3 individual experiments.

Recombinant Tat protein (1 to 300 ng/mL) had no effect on IL-6 release or COX-2 expression by hASMCs and did not affect the responses to OSM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We report here that OSM is an important and unique modulator of human vascular smooth muscle cells through the introduction of specific cytomorphological changes and stimulation of the expression of both IL-6 and COX-2.

OSM induced changes in hASMC morphology with a shift from a spindle-shaped morphology to a polygonal morphology, as characterized by abundant vinculin-rich areas of focal contacts. These morphological changes were associated with an antiproliferative effect of OSM, which was observed at concentrations of 10 and 100 ng/mL. The antimitogenic effect of OSM on hASMCs is in agreement with previous studies performed in human vascular smooth muscle cells,^{5 24} although OSM has been shown to stimulate the growth of rabbit aortic smooth muscle cells²⁴ and AIDS-KS cells.^{5 25} Interestingly, the OSM effects on morphology and proliferation of hASMCs contrast with those reported in endothelial cells, which proliferate and assume a spindle-shaped morphology close to that of KS cells in response to OSM.^{8 26}

OSM stimulated the production of high amounts of IL-6. The release of IL-6 by hASMCs was detected through the use of bioassay as well as specific ELISA, indicating that IL-6 induced by OSM in the supernatants of hASMCs was both specific and bioactive and might act as an unique autocrine and paracrine factor in the vessel wall. The ability of OSM to stimulate IL-6 synthesis by hASMCs is in agreement with previous reports showing that OSM activated endothelial cells to produce IL-6.¹⁰ Modur et al²⁷ recently showed the potency of OSM as a proinflammatory cytokine on the basis of its numerous actions on endothelial cells, including the expression of IL-6, chemokines (IL-8, GRO), and adhesion molecules (selectins, ICAM-1, and VCAM-1). Human fibroblasts and KS cells can also be activated by OSM to produce high amounts of IL-6.²⁵

The kinetics of IL-6 production were striking. The IL-6 production rate was almost constant up to 72 hours after stimulation with OSM, suggesting that IL-6 expression is not downregulated in hASMC monocultures. This is in agreement with observations reported by Loppnow and Libby²⁸ on vascular cell monocultures activated by IL-1ß. The significance of high IL-6 production by hASMCs within the vessel wall is still unclear. Recent findings indicate that IL-6 is involved in angiogenesis, being both a growth factor for vascular cells and a factor of neovascularization for tumors.²⁹ In addition, Modur et al²⁷ recently found an intense staining for OSM in human aortic aneurysms, suggesting a role for this cytokine in chronic vascular inflammation.

OSM also induced the early gene COX-2 and stimulated the release of prostaglandins by hASMCs. OSM treatment led to the marked expression of COX-2 mRNA, COX-2 protein, and COX-2–dependent metabolite accumulation in conditioned medium. Western blot analysis indicated that the COX-2 expression in hASMCs remained elevated after 24 hours of stimulation and declined at 72 hours. Prostaglandin production by OSM-activated hASMCs was enhanced after the addition of exogenous arachidonic acid, indicating an increase in COX-2 activity.³⁰ COX-2 expression represents an early and sensitive response of hASMCs to inflammatory injury, and COX-2–derived metabolites influence many vascular functions, such as vascular tone, thromboresistance, and vascular growth.^{11 31} However, the inhibition of COX-2–dependent prostaglandin release by NS-398 did not unmask any effect of OSM on hASMC proliferation.

Smooth muscle cell activation by OSM was not shared by LIF and was not influenced by LIF in competition studies with OSM. This suggests that the effects of OSM on hASMCs are mediated by the specific OSM receptor. Indeed, OSM is a member of a family of related cytokines that includes LIF and IL-6,¹ and 2 types of OSM receptor complexes have been identified.³² One type of receptor complex, which was recently referred to as type I receptor, is identical to the high-affinity LIF receptor subunit and is composed of a gp130 subunit and the LIF binding subunit gp190. The second type of receptor complex, which was referred to as the type II receptor, is specific for OSM and exhibits no affinity for LIF.³³ In the present study, exogenous recombinant LIF did not affect IL-6 and COX-2 expression on hASMCs, suggesting that the LIF receptor was absent on hASMCs and that OSM acted specifically via the type II receptor. These results are in agreement with findings reported by Brown et al¹⁰ in endothelial cells, showing the presence of a great number of OSM type II receptor on the cell surface.

The signal transduction pathways mediating the secretion of IL-6 and the induction of COX-2 in response to OSM appeared to involve tyrosine kinases, as suggested by the inhibitory effect of genistein, a tyrosine kinase inhibitor. Our results are in agreement with those of Amaral et al,³⁴ who showed that in endothelial cells, OSM uses a tyrosine phosphorylation signal transduction pathway leading to the induction of IL-6 expression. In addition, previous reports showed that COX-2 expression induced by proinflammatory cytokines involves the tyrosine kinase pathway.³⁵ Moreover, the exposure of hASMCs to OSM induced phosphorylation of JAK1 and activation of STAT1, indicating that the JAK/STAT pathway is involved in OSM signaling events in hASMCs. Interestingly, OSM induced a rapid and strong activation of STAT1 but not of STAT3 or STAT5. This specific STAT1 activation by OSM appears to be a feature of the response of hASMCs because in other cell types, IL-6–related cytokines preferentially activate STAT3 and STAT5.³⁶ Experiments in which fludarabine is used as an STAT1 inhibitor²³ indicated that STAT1 activation was directly involved in IL-6 or COX-2 induction by OSM. Our finding that OSM induced a specific IRF1-GAS binding activity in hASMCs, together with the presence of GAS-binding site in the COX-2 gene promoter,³⁷ further suggests that the JAK/STAT pathway mediated OSM-induced COX-2 expression in hASMCs.

Tat protein, an HIV gene product acting as a transactivator for HIV replication, is known to be a potent mitogen for KS cells in cooperation with cytokines⁶ and was therefore tested as an activating factor of hASMCs. We did not observe any stimulation of IL-6 or COX-2 expression by recombinant Tat protein in hASMCs, which is in contrast to Hofman et al,³⁸ who showed a significant increase in IL-6 production induced by Tat in human umbilical vein endothelial cells.

OSM is implicated in a vascular cytokine network, acting in synergy with IL-1. IL-1 is known to be a major stimulus for both IL-6 production and COX-2 expression by vascular smooth muscle cells.^{11 28} In the course of inflammation, multiple cytokines interact in a cascade network. We thus studied the effect of simultaneous incubation of hASMCs with OSM and IL-1ß. We found a striking cooperation between these 2 cytokines to stimulate the expression of IL-6 and COX-2. IL-6 production in the presence of OSM and IL-1 was 2-fold higher than that expected if OSM and IL-1 acted additively. Synergy between OSM and tumor necrosis factor has also been previously observed in endothelial cells.¹⁰ More remarkably, when hASMCs were stimulated with both OSM and IL-1ß, COX-2 expression, which was enhanced compared with stimulation by a single cytokine, remained dramatically elevated even after 72 hours, at a time at which it returned to baseline level in hASMCs activated by only 1 of these cytokines. COX-2 mRNA expression was also increased by the combination of OSM and IL-1 compared with single cytokine activation but in an additive manner. Actinomycin-D almost totally abolished COX-2 expression in cells activated with OSM and IL-1, and mRNA decay time courses suggested that OSM enhanced message stability compared with IL-1 alone. Therefore, the synergistic effect of OSM and IL-1 on COX-2 protein expression most likely involved both transcriptional and post-transcriptional regulations.

In conclusion, OSM can be considered a novel regulator of vascular smooth muscle properties and a potential mediator of vascular inflammation.

	Acknowledgments

 
This work was supported by Grant CSS2 from Agence Nationale pour la Recherche sur le SIDA 1995.

	Footnotes

 
¹ Deceased.

Received July 21, 1999; accepted September 29, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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