Evidence for prostacyclin and cAMP upregulation by bradykinin and insulin-like growth factor 1 in vascular smooth muscle cells

Abstract

Although bradykinin (BK) and insulin like growth factor-1 (IGF-1) have been shown to modulate the functional and structural integrity of the arterial wall, the cellular mechanisms through which this regulation occurs is still undefined. The present study examined the role of second messenger molecules generated by BK and IGF-1 that could ultimately result in proliferative or antiproliferative signals in vascular smooth muscle cells (VSMC). Activation of BK or IGF-1 receptors stimulated the synthesis and release of prostacyclin (PGI(2)) leading to increased production of cAMP in VSMC. Inhibition of p42/p44(mapk) or src kinases prevented the increase in PGI(2) and cAMP observed in response to BK or IGF-1, indicating a role for these kinases in the regulation of cPLA(2) activity in the VSMC. Inhibition of PKC failed to alter production of PGI(2) in response to BK, but further increased both p42/p44(mapk) activation and the synthesis of PGI(2) produced in response to IGF-1. In addition, both BK and IGF-1 significantly induced the expression of c-fos mRNA levels in VSMC, and this effect of BK was accentuated in the presence a cPLA(2) inhibitor. Finally, inhibition of cPLA(2) activity and/or cyclooxygenase activity enhanced the expression of collagen I mRNA levels in response to BK and IGF-1 stimulation. These findings indicate that the effect of BK or IGF-1 to stimulate VSMC growth is an integrated response to the activation of multiple signaling pathways. Thus, the excessive cell growth that occurs in certain forms of vascular disease could reflect dysfunction in one or more of these pathways.

Figure 1. Activation of MAPK by BK and/or IGF-1

VSMC were stimulated with either BK (10⁻⁸ M) and/or IGF-1 (10⁻⁸ M) for 10 min. Cell proteins were separated with SDS-PAGE. MAPK phosphorylation (p42^mapk and p44^mapk) were measured by immunoblot using anti-phosphotyrosine-MAPK antibodies (p-MAPK) and total MAPK was measured in the same immunoblot by stripping the membrane and re-immunoblotting with anti-total MAPK antibodies (t-MAPK). Data are expressed as mean±SE and the bar graphs are representative of 6 separate experiments. * P<0.05 vs. control.

Figure 2. Concentration-dependent effects of BK on PGI₂ (6-keto-PGF_1α) production in VSMC

Serum starved VSMC were stimulated with BK (10⁻¹⁰–10⁻⁶ M) for 10 min., and the release of 6-keto-PGF_1α into the media was measured by RIA. BK produced a concentration dependent increase in 6-keto-PGF_1α with a peak response at 10⁻⁸M. The figure is representative of 8 separate experiments. *P<0.05 vs. basal.

Figure 3. Time-response effects of BK and IGF-1 on PGI₂ (6-keto-PGF_1α) release in VSMC

VSMC were stimulated with either BK (10⁻⁸M) or IGF-1 (10⁻⁸ M) for various times (0–30 min). Both BK and IGF-1 each produced a significant increase in 6-keto-PGF_1α production compared to control values. The peak response was observed 10 min. post stimulation. Figure is representative of 6 separate experiments. *P<0.05 vs. control.

Figure 4. Role of MAPK kinase on MAPK activation (A), the synthesis of PGI₂ (6-keto-PGF_1α, B) and cAMP (C) by BK and IGF-1

VSMC were stimulated with either BK (10⁻⁸M) or IGF-1 (10⁻⁸M) for 10 min in the presence and absence of MEK inhibitor PD98059 (20 μM). MAPK phosphorylation (p42^mapk and p44^mapk) were measured by immunoblot using anti-phosphotyrosine-MAPK antibodies (p-MAPK) and total MAPK was measured in the same immunoblot by stripping the membrane and re-immunoblotting with anti-total MAPK antibodies (t-MAPK). Release of 6-keto-PGF_1α and cAMP into the media, were measured by RIA. Data are expressed as mean±SE and the bar graphs are representative of 6 separate experiments. * P<0.05 vs. control, †P<0.05 vs. BK, #P<0.05 vs. IGF-1.

Figure 5. Role of Src-kinases on MAPK activation (A), the synthesis of PGI₂ (6-keto-PGF_1α, B) and cAMP (C) by BK and IGF-1

VSMC were stimulated with either BK (10⁻⁸M) or IGF-1 (10⁻⁸M) for 10 min in the presence and absence of Src kinase inhibitor PP1 (10 μM). MAPK phosphorylation (p42^mapk and p44^mapk) were measured by immunoblot using anti-phosphotyrosine-MAPK antibodies (p-MAPK) and total MAPK was measured in the same immunoblot by stripping the membrane and re-immunoblotting with anti-total MAPK antibodies (t-MAPK). Release of 6-keto-PGF_1α and cAMP into the media, were measured by RIA. Data are expressed as mean±SE and the bar graphs are representative of 6–8 separate experiments. * P<0.05 vs. control, †P<0.05 vs. BK, #P<0.05 vs. IGF-1.

Figure 6. Role of PKC in IGF-1 induced MAPK phosphorylation and PGI₂ (6-keto-PGF_1α) production

VSMC were stimulated with either BK IGF-1 (10⁻⁸M) for 10 min in the presence and absence of PKC inhibitor bisindolylmaleimide (GFX, 2μM). MAPK phosphorylation (p42^mapk and p44^mapk) were measured by immunoblot using anti-phosphotyrosine-MAPK antibodies (p-MAPK) and total MAPK was measured in the same immunoblot by stripping the membrane and re-immunoblotting with anti-total MAPK antibodies (t-MAPK). Release of 6-keto-PGF_1α into the media, was measured by RIA. Data are expressed as mean±SE and the bar graphs are representative of 5 separate experiments. * P<0.05 vs. control, †P<0.05 vs. IGF-1.

Figure 7. Role of cPLA₂ on IGF-1 and BK-induced c-fos mRNA expression

VSMC were pretreated with AACOCF₃ (20μM), a cPLA2 inhibitor, followed by BK (10⁻⁸M) or IGF-1 (10⁻⁸M) stimulation for 30 min. c-fos and GADPH mRNA levels were measured by Northern blot analysis. The bar graph represents the relative intensities of c-fos mRNA levels/GADPH mRNA levels. Blot shown is representative of at least 5–7 experiments. *P<0.001 vs. control; †P<0.01 vs. BK.

Figure 8. Effect of cyclooxgenase inhibitors on BK-induced and/or IGF-1 induced collagen I mRNA expression

VSMC were pretreated with Indomethacin (Indo 50μM), a cyclooxygenase inhibitor, followed by BK (10⁻⁸M) or IGF-1 (10⁻⁸M) stimulation for 6h. Collagen I and β-actin mRNA levels were measured by real-time PCR. The bar graph represents the relative intensities of collagen I mRNA levels/ β-actin mRNA levels. Blot shown is representative of 3 experiments. *P<0.04 vs. control; †P<0.02 vs. IGF-1.

Figure 9. Scheme depicting BK receptor and IGF-1 receptor signaling through MAPK-dependent pathways

Activation of src-family of tyrosine kinases result in the activation of MAPK which in turn stimulates the activity of cPLA₂. Once activated, cPLA₂ in turn will activate cyclooxygenases (COX) resulting in the production of inhibitory prostaglandins such as PGI_2. PGI₂ in turn will lead to elevated cAMP levels, and consequent attenuation of the responses of BK and IGF-1 to stimulate matrix proteins.