It is well established that hyperplasia and decreased apoptosis of airway smooth muscle cells (ASMCs) play an important role in the asthmatic airway remodeling. Tumor suppressor PTEN gene with phosphatase activity plays an important regulatory role in embryonic development, cell proliferation, and apoptosis, cell cycle regulation, migration (invasion) of the cytoskeleton. We hypotheses that PTEN gene could affect the growth and viability of ASMCs through the regulation of PI3K/Akt, MAPK, and cell cycle-related gene expression. We constructed a recombinant adenovirus to transfect ASMCs. Cells were divided into the overexpression of PTEN gene group (Ad-PTEN-GFP), negative control group (Ad-GFP), and blank control group (DMEM). The cell apoptosis of ASMCs were evaluated by Hoechst-33342 staining and PE-7AAD double-labeled flow cytometry. The cell cycle distribution was observed by flow cytometry with PI staining. The expression of PTEN, p-Akt, total-Akt, p-ERK1/2, total-ERK1/2, cleaved-Caspases-3, Caspases-9, p21, and Cyclin D1 were tested by the Western blotting. Our study revealed that overexpression of PTEN gene did not induce apoptosis of human ASMCs cultured in vitro. However, overexpression of PTEN inhibited proliferation of human ASMCs cultured in vitro and was associated with downregulation of Akt phosphorylation levels, while did not affect ERK1/2 phosphorylation levels. Moreover, overexpression of PTEN could induce ASMCs arrested in the G0/G1 phase through the downregulation of Cyclin D1 and upregulation of p21 expressions.
Cell adhesion to the extracellular matrix is required to execute growth factor (GF)-mediated cell behaviors, such as proliferation. A major underlying mechanism is that cell adhesion enhances GF-mediated intracellular signals, such as extracellular signal-regulated kinase (Erk). However, because GFs use distinct mechanisms to activate Ras-Erk signaling, it is unclear whether adhesion-mediated enhancement of Erk signaling is universal to all GFs. We examined this issue by quantifying the dynamics of Erk signaling induced by epidermal growth factor, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) in NIH-3T3 fibroblasts. Adhesion to fibronectin-coated surfaces enhances Erk signaling elicited by epidermal growth factor but not by bFGF or PDGF. Unexpectedly, adhesion is not always a positive influence on GF-mediated signaling. At critical subsaturating doses of PDGF or bFGF, cell adhesion ablates Erk signaling; that is, adhesion desensitizes the cell to GF stimulation, rendering the signaling pathway unresponsive to GF. Interestingly, the timing of growth factor stimulation proved critical to the desensitization process. Erk activation significantly improved only when pre-exposure to adhesion was completely eliminated; thus, concurrent stimulation by GF and adhesion was able to partially rescue adhesionmediated desensitization of PDGF-and bFGF-mediated Erk and Akt signaling. These findings suggest that adhesion-mediated desensitization occurs with rapid kinetics and targets a regulatory point upstream of Ras and proximal to GF receptor activation. Thus, adhesion-dependent Erk signaling is not universal to all GFs but, rather, is GF-specific with quantitative features that depend strongly on the dose and timing of GF exposure.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.