| Summary: | 博士 === 臺北醫學大學 === 臨床醫學研究所 === 97 === The aim of this thesis is to investigate the responses of vascular cells,
including endothelial cells (ECs) and smooth muscle cells (VSMCs), to
acute hypoxia and the underlying molecular mechanisms. Discoidin
domain receptor-2 (DDR2) is a receptor tyrosine kinase that binds to the
extracellular matrix. We first investigated the role of hypoxia in DDR2
expression in VSMCs and the underlying mechanism. Subjecting VSMCs
to hypoxia induced DDR2 expression; treatments with a specific inhibitor
(SB203580) of p38 mitogen-activated protein kinase (MAPK) or
p38-specific small interference RNA (siRNA) abolished this
hypoxia-induced DDR2 expression. Gel shifting assays showed that
hypoxia increased the Myc–Max–DNA binding activity in the promoter
region of DDR2; inhibition of p38 MAPK activation by SB203580 and
p38-specific siRNA blocked hypoxia-induced DDR2 promoter activity.
Hypoxia also induced matrix metalloproteinase-2 (MMP-2) activity in
VSMCs and increased their migration. These VSMC responses to
hypoxia were inhibited by DDR2- and p38-specific siRNAs. Our results
suggested that hypoxia induces DDR2 expression in VSMCs at the
transcriptional level, which is mediated by the p38 MAPK pathway and
contributes to VSMC migration.Since activating transcription factor 3 (ATF3), a stress-inducible
transcription factor playing significant roles in cellular responses to stress,
including hypoxia, we further investigated the role of ATF3 in cellular
responses to hypoxia. Vascular ECs were subjected to acute hypoxia and
ATF3 expression was examined. ECs exposed to hypoxia transiently
induced ATF3 expression. A transient increase in the activation of
c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein
kinase (MAPK) in ECs was observed; however, only ECs pretreated with
a specific inhibitor to JNK suppressed the hypoxia-induced ATF3
expression. ECs exposed to acute hypoxia transiently increased
endothelial nitric oxide synthase (eNOS) activity. Pre-treating ECs with a
specific inhibitor to eNOS (L-NAME) or PI3-kinase significantly
inhibited the hypoxia-induced JNK activation and ATF3 expression.
ATF3 induction has been shown to inhibit matrix metalloproteinase-2
(MMP-2) expression. Consistently, ECs exposed to hypoxia attenuated
the MMP-2 expression. These results suggest that the ATF3 induction by
acute hypoxia is mediated by nitric oxide and the JNK pathway in ECs.
Recent studies indicated that protein S-nitrosylation (S-NO) plays an
important role in regulating proteins’ function and thus is of significance
in cell physiology. We further investigated the effect of hypoxia on EC
S-NO. A modified biotin-switch method coupled with proteomic
approach demonstrated that at least 11 major proteins have significant increase in S-NO in ECs after acute hypoxia. Mass analysis by
CapLC/Q-TOF identified those as Ras-GTPase-activating protein, protein
disulfide-isomerase, human elongation factor-1-delta, tyrosine
3/tryptophan 5-monooxygenase activating protein, and several
cytoskeleton proteins. Further understanding of the functional relevance
of these S-nitrosylated proteins and the previous findings of DDR2 and
ATF3 activities may provide a molecular basis for understanding the
ischemia-induced vascular physiologies.
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