UVM Theses and Dissertations
Format:
Print
Author:
Wang, Hsiang-Ming
Dept./Program:
Cell and Molecular Biology Program
Year:
2005
Degree:
Ph. D.
Abstract:
Ultraviolet (UV) irradiation is a potent stress that damages DNA and triggers programmed cell death. UV also has been shown to activate growth factor receptors and transduce signals through mitogen activated protein kinase (MAPK) cascades, suggesting that early signals for UV-induced MAPK activation are generated at the plasma membrane. Reports differ however, with respect to the involvement of different growth factor receptors and their downstream signaling molecules. We investigated the role of insulin receptors in mediating the response to UV irradiation. By western blotting and confocal microscopy of the insulin receptor (IR), we show that while UVC does not induce receptor tyrosine phosphorylation, it rapidly promotes receptor redistribution and internalization which may serve to stimulate downstream signaling.
In the absence of exogenous growth factors, UVC irradiation of CHO cells overexpressing the human IR (CHO/hIR) activated anti-apoptotic signaling requiring phosphatidylinositol-3-kinase (PI3K) but not Erk or p38 MAPK. Using thin layer chromatography, we observed that PI3K is modestly stimulated by UVC in CHO/hIR and parental CHO cell lines, yet the basal activity is higher in CHO/hIR. UVC also stimulates Akt phosphorylation more potently in CHO/hIR and this correlates with mitochondrial protection as determined by the mitochondrial potential sensitive dye JC-1. In conclusion, UVC-irradiation induces anti-apoptotic signaling involving redistribution of the IR and PI3K-regulated Akt activation, inhibiting the mitochondrial apoptotic pathway.
We also explored the upstream components involved in UV activation of the p38 pathway. Using in vitro kinase assay, we found that PI3K is necessary but not sufficient for UV induced p38 activation. Since PI3K interacts with Ras and Rac, we showed that constitutively active Ras or Rac activates p38 in the presence of UV, while dominant negative Ras and Rac decreased p38 activity, indicating Ras and Rac are also necessary but not sufficient for p38 activation. By co-transfection of RasPI3K or RacPI3K, we demonstrated a Ras-PI3K-Rac pathway for UV induction of p38. Since constitutively active forms of Ras, Rac, and PI3K failed to activate p38 in the absence of UV, an additional UV-induced pathway is required for full p38 activation.
UV-induced DNA damage in eukaryotes can only be repaired with Nucleotide Excision Repair (NER). It has been shown that upon UV irradiation, NER is activated while transcription is reduced. The involvement of Transcription Factor IIH (TFIIH) in both transcription and NER suggests a potential role of regulating this transition. UV also strongly activates pro-apoptotic kinases JNK and p38. Since both CDK7 and p62 undergo phosphorylation upon TFIIH regulated mitotic inhibition, and CDK7 contains a putative MAPK consensus sequence, we examined potential phosphorylation of TFIIH by JNK or p38 as a way to regulate transcription/repair.
HeLa cells were transfected with epitope-tagged JNK, ERK or p38. After UV treatment, these MAPKs were irnrnuno-precipitated and in vitro kinase assays were performed using purified subunits of TFIIH including CDK7, p62 and p89. However, none of the above tested proteins were directly phosphorylated by the three MAP kinases, indicating that the role of JNK and p38 in repair is not directly through phosphorylation of TFIIH. In conclusion, we demonstrated that W irradiation activates both pro- and antiapoptotic pathways using distinct signaling components.
In the absence of exogenous growth factors, UVC irradiation of CHO cells overexpressing the human IR (CHO/hIR) activated anti-apoptotic signaling requiring phosphatidylinositol-3-kinase (PI3K) but not Erk or p38 MAPK. Using thin layer chromatography, we observed that PI3K is modestly stimulated by UVC in CHO/hIR and parental CHO cell lines, yet the basal activity is higher in CHO/hIR. UVC also stimulates Akt phosphorylation more potently in CHO/hIR and this correlates with mitochondrial protection as determined by the mitochondrial potential sensitive dye JC-1. In conclusion, UVC-irradiation induces anti-apoptotic signaling involving redistribution of the IR and PI3K-regulated Akt activation, inhibiting the mitochondrial apoptotic pathway.
We also explored the upstream components involved in UV activation of the p38 pathway. Using in vitro kinase assay, we found that PI3K is necessary but not sufficient for UV induced p38 activation. Since PI3K interacts with Ras and Rac, we showed that constitutively active Ras or Rac activates p38 in the presence of UV, while dominant negative Ras and Rac decreased p38 activity, indicating Ras and Rac are also necessary but not sufficient for p38 activation. By co-transfection of RasPI3K or RacPI3K, we demonstrated a Ras-PI3K-Rac pathway for UV induction of p38. Since constitutively active forms of Ras, Rac, and PI3K failed to activate p38 in the absence of UV, an additional UV-induced pathway is required for full p38 activation.
UV-induced DNA damage in eukaryotes can only be repaired with Nucleotide Excision Repair (NER). It has been shown that upon UV irradiation, NER is activated while transcription is reduced. The involvement of Transcription Factor IIH (TFIIH) in both transcription and NER suggests a potential role of regulating this transition. UV also strongly activates pro-apoptotic kinases JNK and p38. Since both CDK7 and p62 undergo phosphorylation upon TFIIH regulated mitotic inhibition, and CDK7 contains a putative MAPK consensus sequence, we examined potential phosphorylation of TFIIH by JNK or p38 as a way to regulate transcription/repair.
HeLa cells were transfected with epitope-tagged JNK, ERK or p38. After UV treatment, these MAPKs were irnrnuno-precipitated and in vitro kinase assays were performed using purified subunits of TFIIH including CDK7, p62 and p89. However, none of the above tested proteins were directly phosphorylated by the three MAP kinases, indicating that the role of JNK and p38 in repair is not directly through phosphorylation of TFIIH. In conclusion, we demonstrated that W irradiation activates both pro- and antiapoptotic pathways using distinct signaling components.