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Arterial smooth muscle cell (SMC) phenotype and proliferation is regulated by their surrounding collagens, which transform from fibrillar to monomeric type in atherogenesis, and platelet-derived growth factor (PDGF)-BB/interleukin (IL)-1β. This study aims at elucidating the mechanisms by which physical (monomeric vs . fibrillar collagens) and chemical (PDGF-BB/IL-1β vs . vehicle controls) stimuli modulate SMC cycle and proliferation. SMCs were cultured on monomeric vs . fibrillar type I collagens. In parallel experiments, SMCs on fibrillar collagen were co-stimulated with PDGF-BB/IL-1β. These physical and chemical factors induced common SMC cycle signaling events, including up-regulations of cyclin-dependent kinase-4/6 and cyclins A/D1, phosphorylation of retinoblastoma (Rb) and its dissociations with E2F2/3. The physical and chemical inductions of SMC cycle signaling and progression were oppositely regulated by phosphatidylinositol 3-kinase (PI3K)-mediated Akt and p38 mitogen-activated protein kinase (MAPK). Fibrillar collagen degraded p66Shc, whose Ser36-phosphorylation plays important roles in the modulation of SMC cycle. Monomeric collagen and PDGF-BB/IL-1β co-stimulation induced p66Shc expression and Ser36-phosphorylation through β 1 integrin and PDGF receptor-β, respectively. In conclusion, our results demonstrate that fibrillar collagen-regulated p66Shc converges the physical and chemical stimuli to modulate SMC cycle and proliferation through PI3K-mediated Akt and p38 MAPK and their opposite regulation in downstream common cell cycle signaling cascades.

Human telomerase reverse transcriptase (hTERT) and oncogene c-Myc have been shown to regulate cell proliferation. Our previous studies demonstrated that fibrillar collagen mediates vascular smooth muscle cell (SMC) cycle progression and proliferation in response to platelet-derived growth factor (PDGF)-BB and interleukin (IL)-1β. However, whether hTERT and c-Myc are involved in these fibrillar collagen-mediated SMC responses remain unclear. The present study elucidated the regulatory role of hTERT and c-Myc in PDGF-BB/IL-1β-induced cell cycle progression in SMCs on fibrillar collagen and its underlying mechanisms. Our results showed that PDGF-BB and IL-1β exert synergistic effects to induce hTERT expression, but not its activity, in human arterial SMCs on fibrillar collagen. This PDGF-BB/IL-1β-induced up-regulation of hTERT contributes to cell cycle progression in SMCs through the up-regulation of cyclin-dependent kinase-6 and down-regulations of p27(KIP1) and p21(CIP1). In addition, PDGF-BB/IL-1β induces up-regulation of c-Myc in SMCs on fibrillar collagen; this response is mediated by the increased binding of hTERT, which can form complexes with TPP1 and hnRNPK, to the guanine-rich region of the c-Myc promoter and consequently contributes to cell cycle progression in SMCs on fibrillar collagen. Moreover, the PDGF-BB/IL-1β-induced hTERT and c-Myc expressions are regulated by phosphatidylinositol 3-kinase/Akt in SMCs on fibrillar collagen. Our findings provide insights into the mechanisms by which hTERT and c-Myc regulates SMC cell cycle progression and proliferation on fibrillar collagen in response to chemical stimuli.

Chitooligosaccharides (COS) have been shown to regulate various cellular and biological functions. However, the effect of COS on inflammatory responses of the cells remains unclear. We investigated the regulatory effect of highly N-acetylated COS (NACOS) on tumor necrosis factor-alpha (TNF-alpha)-induced endothelial cell (EC) E-selectin expression, which is crucial for leukocyte recruitment. ECs were kept as controls or pre-treated with NACOS for different times, and then stimulated with TNF-alpha for 4h. The results show that pre-treating ECs with NACOS inhibited the TNF-alpha-induced E-selectin expression in a dose- and time-dependent manner. This NACOS-mediated inhibition in E-selectin expression was regulated at the transcriptional level, but not due to changes in mRNA stability. Stimulation of ECs with TNF-alpha-induced rapid increases in the phosphorylation of their mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinase (ERK), c-Jun-NH2-terminal kinase (JNK), and p38 MAPK]; the inhibitor for JNK (i.e., SP600125), but not those for ERK (i.e., PD98059) and p38 MAPK (i.e., SB203580), attenuated this TNF-alpha-induced E-selectin expression. Pre-treating ECs with NACOS inhibited the TNF-alpha-induced JNK activation, suggesting that JNK was involved in the inhibitory effect of NACOS on TNF-alpha-induced E-selectin expression. Pre-treating ECs with NACOS inhibited the TNF-alpha-induced p65 and p50 mRNA expressions. Gel shifting and chromatin immunoprecipitation assays showed that NACOS blocked the TNF-alpha-induced increases in the binding activity and in vivo promoter binding of nuclear factor-kappaB (NF-kappaB) in ECs. Our findings provide a molecular mechanism by which NACOS inhibit TNF-alpha-induced E-selectin expression in ECs, and a basis for using NACOS in pharmaceutical therapy against inflammation.