MHC class I and integrin ligation induce ERK activation via an mTORC2-dependent pathway

Abstract

The aim of this study was to characterize the interaction between mTOR and ERK in primary endothelial cells (EC) following MHC class I and integrin ligation. Ligation of MHC class I molecules or integrins on the surface of EC leads to phosphorylation of ERK at Thr202/Tyr204. We utilized small interfering RNA (siRNA) blockade of mTOR and proteins involved in mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) to define a relationship between mTOR and ERK following MHC class I signaling. We found mTORC2 was responsible for MHC class I and integrin induced phosphorylation of ERK at Thr202/Tyr204. We corroborated these results demonstrating that long-term exposure to rapamycin also inhibited ERK pathway activation in response to MHC class I signaling. Our results demonstrate, for the first time, that engagement of either MHC class I or integrin on the surface of EC leads to ERK activation through an mTORC2-dependent pathway. KeywordsMHC; HLA class I; ERK; mTORC2; siRNA; Endothelial cells; Signal transduction mTOR plays an essential role in integrin and MHC class I-induced cell proliferation and survival through formation of two distinct multi-protein molecular complexes, mTORC1 and mTORC2 [1][2][3][4]. mTORC1 consists of mTOR in association with regulatory associated protein of mTOR (Raptor) and GβL [5]. mTORC1 phosphorylates p70 S6 Kinase and eukaryotic initiation factor 4E binding protein 1 which activate translational machinery to increase cell proliferation [6,7]. mTORC2 is composed of mTOR binding to rapamycin insensitive companion of mTOR (Rictor), GβL, stress-activated protein kinase-interacting protein 1 (Sin1) and the newly discovered associations with PRoline-Rich protein 5 (PRR5) or the synonymous protein observed with Rictor 1 (Protor-1), and P-REX1 [8][9][10][11][12]. mTORC2 activates Akt at Ser473, Rac GTPase, cell migration and effects reorganization of the actin cytoskeleton [11][12][13][14]. Recent work utilizing mouse knockout models emphasizes the importance of mTOR for growth and proliferation in development [15,16]. Homozygous mTOR (−/−) mice have * Corresponding author: Fax: + 1 310 206 3216; Email address: ereed@mednet.ucla.edu (E. F. Reed). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [17,18]. These data show both mTOR complexes are involved in signal transduction leading to cell proliferation, however, the mechanism(s) by which mTORC2 participates in mitogenic signaling remains less well defined. NIH Public AccessERK1/2 are members of a family of serine/threonine kinases activated by a variety of growth factors, cell surface receptors, int...

Report of the Inefficacy of Eculizumab in Two Cases of Severe Antibody-Mediated Rejection of Renal Grafts

“…Direct endothelial cell activation by DSAs may also contribute to microvascular damage. Thus, it was shown that antibodies binding to class I HLA molecules expressed at the surface of endothelial cells from cardiac grafts may transduce intracellular pathways, activate cells, and eventually induce proliferation, suggesting that alloantibodies may directly contribute to chronic transplant vasculopathy (14,15). Furthermore, Naemi et al (16) demonstrated that HLA class-I antibodies modulate endothelium-leukocyte interactions by up-regulating chemokine and integrin expressions by the activated endothelium, leading to more potential of leukocyte recruitment and binding, and hence allograft inflammation.…”

HLA class I-mediated stress fiber formation requires ERK1/2 activation in the absence of an increase in intracellular Ca2+ in human aortic endothelial cells

“…The hybridoma (HB-95) W6/32, recognizing a monomorphic epitope on HLA class I, was purchased from the American Type Culture Collection. W6/32 was added to ECs for a final concentration of 0.1 g/ml, which was previously determined to be optimal for examining ERK1/2 phosphorylation (26). The mouse IgG monoclonal antibody isotype control was purchased from SigmaAldrich.…”

Mechanisms of human smooth muscle cell proliferation and transplant vasculopathy induced by HLA class I antibodies: In vitro and in vivo studies

“…Furthermore, they induce ERK phosphorylation by an mTOR-dependent mechanism [25,26]. mTOR also appears to be involved in the phosphorylation of Akt and in the proliferative effect of anti-HLA antibodies on EC, but its activation is dependent on Src and FAK [25,26]. Upstream of this signaling, the interaction between HLA class I and integrin b4 is essential for the phosphorylation of these proteins and the proliferation of EC in response to anti-HLA antibodies [21].…”

“…In addition, it was shown that the PI3K/Akt pathway was activated by Src and FAK, leading to increased expression of pro-survival proteins Bcl-2 and Bcl-xL [23] and of antioxidant defense such as HO-1 [24] in EC stimulated by anti HLA antibodies. Furthermore, they induce ERK phosphorylation by an mTOR-dependent mechanism [25,26]. mTOR also appears to be involved in the phosphorylation of Akt and in the proliferative effect of anti-HLA antibodies on EC, but its activation is dependent on Src and FAK [25,26].…”