Phosphatidylinositol 3-kinase/Akt pathway is involved in transforming growth factor-β1-induced phenotypic modulation of 10T1/2 cells to smooth muscle cells
Introduction
Atherosclerotic lesions are characterized by disturbances in cell differentiation, including the appearance of immature smooth muscle cells (SMCs) [1]. The presence of immature SMCs in atherosclerotic lesions may be a result of recapitulation of embryonic mechanisms in the artery wall [1]. Several cell types present in the artery are potential candidates as precursors of SMCs, which appear to originate from the lateral mesenchyme (i.e., mesoderm), under the influence of various factors [2], [3]. Among these factors, transforming growth factor-β1 (TGF-β1) is well known to induce differentiation of mesenchymal cells toward a SMC phenotype, and an increased expression of TGF-β1 associated with active mesenchymal cells has been shown to occur in human atherosclerotic plaques [3], [4]. In the mouse 10T1/2 multipotent mesenchymal cell line, TGF-β1 stimulates cell growth with the up-regulation of several SMC differentiation markers, such as smooth muscle α-actin (SMα-actin), smooth muscle myosin heavy chain (SM-MHC), smooth muscle protein 22-α (SM22α), and calponin [1], [2], [5]. Thus, it is important to clarify the mechanism responsible for the phenotypic modulation of mesenchymal cells to SMCs upon TGF-β1 stimulation.
Many extracellular stimuli elicit specific biologic responses through activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt cascades [6], [7]. Three subgroups of mammalian MAPKs have been molecularly characterized: extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun-NH2-terminal kinase (JNK). While ERK is activated by mitogenic stimuli and plays a key role in cell proliferation and differentiation [8], JNK and p38 MAPK are activated by environmental stresses such as heat shock, ultraviolet irradiation, and inflammatory cytokines, and play important roles in apoptosis and cytokine expression [9], [10], [11], [12]. In addition, the PI3K/Akt pathway has been shown to regulate a number of cellular processes including cell cycle progression, glucose metabolism, angiogenesis, cell motility, and apoptosis [13]. Thus, the PI3K/Akt and the MAPK superfamily regulate a variety of cellular functions; however, the role of these signaling pathways in the TGF-β1-induced mesenchymal differentiation to SMCs has not been determined.
In the present study, we investigated the roles of ERK, JNK, p38 MAPK, and PI3K/Akt in inducing the SMC phenotype characterized by the expressions of SMα-actin, SM-MHC, and SM22α in TGF-β1-stimulated 10T1/2 mesenchymal cells. To this end, we examined the expressions of these SMC differentiation markers and the activation of ERK, JNK, p38 MAPK, and Akt in TGF-β1-stimulated 10T1/2 cells, and the effects of specific inhibitors of these signaling pathways, i.e., LY294002 for PI3K/Akt [14], SP600125 for JNK [15], SB203580 for p38 MAPK [16], and PD98059 for MAPK-1 (MEK-1), which is upstream of ERK [17], on the TGF-β1-induction of SMC differentiation markers in 10T1/2 cells.
Section snippets
Materials
The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): mouse monoclonal antibody (sc-1647) against ERK2; mouse monoclonal Phospho-ERK antibody (sc-7383) detecting phosphorylation at the Tyr-204 site of ERK1/2; mouse monoclonal antibody (sc-7345) against JNK2; mouse monoclonal Phospho-JNK antibody (sc-6254) detecting phosphorylation at the Thr-183 and Tyr-185 sites of JNK; and rabbit polyclonal antibody against serum response factor (SRF). The following
TGF-β1 induces SMα-actin and SM-MHC expressions in 10T1/2 cells in a time-dependent and a dose-independent manner
We first examined the time course of the effects of TGF-β1 on the SMα-actin and SM-MHC expressions in 10T1/2 cells. To this end, we stimulated 10T1/2 cells with 2 ng/ml of TGF-β1 and immunoblotted the cell lysates at 0.5, 2, 8, 16, and 24 h after cultivation. Analysis of temporal expressions showed that SMα-actin and SM-MHC protein levels were induced by TGF-β1 at 8 h and remained elevated for 24 h (Fig. 1A). Northern blot analysis also revealed this time-dependent increase in the levels of
Discussion
In the present study, we analyzed the roles of ERK, JNK, p38 MAPK, and PI3K/Akt in mediating the TGF-β1-stimulated transformation of 10T1/2 mesenchymal cells into SMC phenotype, characterized by the expressions of differentiation markers, which included SMα-actin, SM-MHC, and SM22α. We concluded that the PI3K/Akt pathway, but not ERK, JNK, and p38 MAPK, is involved in the TGF-β1-mediated phenotypic changes of 10T1/2 cells to SMCs based on several lines of evidence. First, TGF-β1 induced an
Acknowledgments
This work was supported by grants ME-094-PP-06 (Jeng-Jiann Chiu) from the National Health Research Institutes, 94-3112-B-400-005 and 94-2321-B-400-002 from the National Science Council, Taiwan, ROC, and HL19454 (Shu Chien) from the National Heart, Lung, and Blood Institute, USA. The work was conducted in part in affiliation with the Center of Tissue Engineering and Stem Cell Research, National Chung-Hsing University, Taichung, Taiwan, ROC.
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