Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor

Abstract

Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8–37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8–37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [{N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride}] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin {[1S-(1α,6bα,9aβ,11α,11bβ)]-11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione}, a PI3-kinase inhibitor, attenuated only calcitonin gene-related peptide-induced ERK and not P38 MAPK activation. Thus, these data suggest that activation of ERK by calcitonin gene-related peptide involves a H89-sensitive protein kinase A and a wortmannin-sensitive PI3-kinase while activation of p38 MAPK by calcitonin gene-related peptide involves only the H89 sensitive pathway and is independent of PI3 kinase. This also suggests that although both ERK and P38 can be activated by protein kinase A, the distal signaling components to protein kinase A in the activation of these two kinases (ERK and P38) are different.

Introduction

Mitogen-activated protein kinases (MAPK's) are proline directed serine/threonine kinases that are activated in response to a variety of extracellular stimuli. There are five distinct groups of MAPK's in mammalian cells. At present, the best characterized are extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38 MAPK) (Denhardt, 1996). Activation of growth factor receptors, G-protein-coupled receptors, inflammatory cytokine receptors and induction of stress by ultraviolet light or high osmolarity have been shown to activate one or more of these MAPK pathways resulting in varied biological responses including cell proliferation, cell death, matrix production, secretion etc. Each of these pathways prototypically consists of a small G-protein (e.g., ras) activating a mitogen-activated protein kinase kinase kinase (MAPKKK) or MEKK (e.g., raf). Activated MEKK phosphorylates a mitogen-activated protein kinase kinase (MAPKK or MEK) (e.g., MEK1/2) and activates it. MEK is a dual function kinase that can phosphorylate threonine and tyrosine residues. When activated, MEK phosphorylates and activates a MAPK (e.g., ERK, JNK, P38 MAPK) Denhardt, 1996, Neary, 1997, Robinson and Cobb, 1997. Activation of MAPK plays important roles in integrating the effects of extracellular signals on multiple cellular functions, including differentiation, proliferation and transformation.

Calcitonin gene-related peptide is a neuropeptide with diverse biological effects including potent vasodilator activity DeFeudis, 1992, Poyner, 1995, Brain and Cambridge, 1996. The receptor for calcitonin gene-related peptide has been cloned from various species including human, rat and pig (Wimalawansa, 1996). The receptor displays 7 transmembrane domains and shows significant homology with a subfamily of G-protein-coupled receptors that includes calcitonin, vasoactive intestinal peptide, secretin, glucagon and corticotropin releasing factor. Messenger RNA encoding the calcitonin gene-related peptide receptor is expressed in relatively high levels in human heart and lung (Wimalawansa, 1996). Expression of the recombinant calcitonin gene-related peptide receptor in stably transfected human embryonic kidney (HEK) 293 cells has enabled study of their ligand-stimulated signal transduction. Like other receptors of this family, the recombinant calcitonin gene-related peptide receptor was shown to be capable of activating adenylyl cyclase (Aiyar et al., 1996), as well as rapid increase of intracellular Ca²⁺ through the activation of phospholipase C (Aiyar et al., 1999). The purpose of this study was to delineate the signaling events downstream of cAMP in 293 cells by calcitonin gene-related peptide. Here we show that the recombinant calcitonin gene-related peptide receptor expressed in HEK-293 cells respond to calcitonin gene-related peptide stimulation with increased ERK and P38 activity and that this effect is mediated by the calcitonin gene-related peptide type 1 receptor in a cAMP-dependent manner.