Xiaomei Qi

p38 Isoforms Have Opposite Effects on AP-1-dependent Transcription through Regulation of c-Jun THE DETERMINANT ROLE OF THE ISOFORMS IN THE p38 MAPK SIGNAL SPECIFICITY

p38 MAPK pathway signaling is known to participate in cell proliferation, apoptosis, and differentiation, in a manner dependent on the cellular context. The factors that determine the specific biological response in a given cell type, however, remain largely unknown. We report opposite effects of the p38 isoforms on regulation of AP-1-dependent activities by p38 activators MAPK kinase 6 (MKK6) and/or arsenite in human breast cancer cells. The p38β isoform increases the activation of AP-1 transcriptional activities by MKK6 and/or arsenite, whereas p38γ/p38δ inhibits or has no effect on the stimulation. The p38β does so by increasing the levels of phosphorylated c-Jun, whereas the p38γ and -δ isoforms may act by regulating the c-jun transcription. AP-1-dependent processes such as vitamin D receptor gene promoter activation and cellular proliferation were similarly activated by the p38β or inhibited by the p38γ and/or -δ isoforms. Whereas the human breast cancer cells express all four isoforms, mouse NIH 3T3 and EMT-6 cells express only some of the p38 family members, with p38β higher in 3T3 cells but p38δ only detected in the EMT-6 line. Consistent with the positive and negative roles of p38β and p38δ in AP-1 regulation, MKK6 stimulates AP-1-dependent transcription in NIH 3T3 but not EMT-6 cells. In support of a role of c-Jun regulation by p38 isoforms in determining AP-1 activity, the levels of endogenous c-Jun and its phosphorylated form on p38 activation are higher in NIH 3T3 cells. These results demonstrate the contrasting activities of the different p38 isoforms in transmitting the upstream signal to AP-1 and show that the expression profile of p38 isoforms determines whether the p38 signal pathway activates or inhibits AP-1-dependent processes.

c-Fos Degradation by the Proteasome AN EARLY, Bcl-2-REGULATED STEP IN APOPTOSIS

c-Fos is a transcription factor that promotes cell growth, differentiation, and transformation. We found that c-Fos was degraded when WEHI7.2 mouse lymphoma cells were induced to undergo apoptosis with the calcium ATPase inhibitor, thapsigargin, or the glucocorticoid hormone, dexamethasone. The degradation of c-Fos preceded caspase-3 activation and apoptotic nuclear chromatin condensation and was inhibited by the proteasome inhibitors MG132,N-acetyl-leucyl-leucyl-norleucinal, and lactacystin. Stable transfection of WEHI7.2 cells with a mutant form of c-Fos that was not degraded by the proteasome inhibited apoptosis. Also, overexpression of Bcl-2 in WEHI7.2 cells blocked c-Fos degradation and inhibited apoptosis. The results indicate that proteasome-mediated degradation of c-Fos is an early, Bcl-2-regulated step in apoptosis induction by thapsigargin and dexamethasone. These findings suggest that c-Fos may have a protective action that is eliminated by proteasome-mediated degradation and preserved by Bcl-2.

p38γ MAPK Cooperates with c-Jun in trans-Activating Matrix Metalloproteinase 9

Mitogen-activated protein kinases (MAPKs) regulate gene expression through transcription factors. However, the precise mechanisms in this critical signal event are largely unknown. Here, we show that the transcription factor c-Jun is activated by p38γ MAPK, and the activated c-Jun then recruits p38γ as a cofactor into the matrix metalloproteinase 9 (MMP9) promoter to induce its trans-activation and cell invasion. This signaling event was initiated by hyperexpressed p38γ that led to increased c-Jun synthesis, MMP9 transcription, and MMP9-dependent invasion through p38γ interacting with c-Jun. p38γ requires phosphorylation and its C terminus to bind c-Jun, whereas both c-Jun and p38γ are required for the trans-activation of MMP9. The active p38γ/c-Jun/MMP9 pathway also exists in human colon cancer, and there is a coupling of increased p38γ and MMP9 expression in the primary tissues. These results reveal a new paradigm in which a MAPK acts both as an activator and a cofactor of a transcription factor to regulate gene expression leading to an invasive response.

p38 MAPK activation selectively induces cell death in K-ras-mutated human colon cancer cells through regulation of vitamin D receptor.

ras is the most characterized oncogene in human cancer, and yet there are no effective therapeutics to selectively target this oncogene. Our previous work demonstrated the inhibitory activity of the p38 pathway in Ras proliferative signaling in experimental NIH 3T3 cells (Chen, G., Hitomi, M., Han, J., and Stacey, D. W. (2000) J. Biol. Chem. 275, 38973-38980). Here we explore the therapeutic potential of p38 kinase activation in human colon cancer cells with and without endogenous K-ras activation. p38 activation by both adenovirus-mediated gene delivery of constitutively active p38 activator MKK6 and by arsenite selectively induces cell death in K-ras-activated human colon cancer HCT116 cells but not in the K-ras-disrupted HCT116-derived sublines. The cell death-inducing effect of MKK6 is not because of its selective activation of p38 kinase or its downstream transcription factor substrates, ATF-2 or c-Jun, in K-ras-activated cells. Rather, cell death in K-ras-activated cells is linked to the down-regulation of vitamin D receptor (VDR) by an AP-1-dependent mechanism. Forced VDR expression in K-ras-activated cells inhibits p38 activation-induced cell death, and inhibition of endogenous VDR protein expression in K-ras-disrupted cells increased the arsenite-induced toxicity. Analysis of an additional two human colon cancer cell lines with and without K-ras mutation also showed a K-ras- and VDR-dependent toxicity of MKK6. Hence, p38 pathway activation selectively induces cell death in K-ras-mutated human colon cancer cells by mechanisms involving the suppression of VDR activity.

Essential role of p38γ in K-Ras transformation independent of phosphorylation

MAPK cascades play the critical role in regulating Ras oncogene activity by phosphorylation-dependent mechanisms. Whereas the ERK MAPK pathway is required for Ras transformation, our previous works established that the p38 activity is inhibitory to Ras signaling in both experimental and ras-mutated cancer cells (Chen, G., Hitomi, M., Han, J., and Stacey, D. W. (2000) J. Biol. Chem. 275, 38973-38980; Qi, X., Tang, J., Pramanik, R., Schultz, R. M., Shirasawa, S., Sasazuki, T., Han, J., and Chen, G. (2004) J. Biol. Chem., 279, 22138-22144). Here we report that K-Ras activated p38γ, a p38 MAPK family member, by inducing its expression without increasing its phosphorylation and that depletion of induced p38γ suppressed Ras transformation in rat intestinal epithelial cells. This p38γ activity contrasts with that of its family member, p38α, which is activated by Ras through phosphorylation, leading to an inhibition of Ras transformation. Mechanistic analyses showed that unphosphorylated p38γ may promote Ras transformation through an increased complex formation with ERK proteins. Significantly, functional p38γ protein was expressed only in K-ras-mutated human colon cancer cells, and p38γ transcripts were ubiquitously increased in a set of primary human colon cancer tissues. These studies thus demonstrate the essential role of p38γ in K-Ras transformation independent of phosphorylation, and elevated p38γ may serve as a novel diagnostic marker and therapeutic target for human colon cancer.

PTPH1 Dephosphorylates and Cooperates with p38γ MAPK to Increase Ras Oncogenesis through PDZ-Mediated Interaction

Protein phosphatases are believed to coordinate with kinases to execute biological functions, but examples of such integrated activities, however, are still missing. In this report, we have identified protein tyrosine phosphatase H1 (PTPH1) as a specific phosphatase for p38gamma mitogen-activated protein kinase (MAPK) and shown their cooperative oncogenic activity through direct binding. p38gamma, a Ras effector known to act independent of its phosphorylation, was first shown to require its unique PDZ-binding motif to increase Ras transformation. Yeast two-hybrid screening and in vitro and in vivo analyses further identified PTPH1 as a specific p38gamma phosphatase through PDZ-mediated binding. Additional experiments showed that PTPH1 itself plays a role in Ras-dependent malignant growth in vitro and/or in mice by a mechanism depending on its p38gamma-binding activity. Moreover, Ras increases both p38gamma and PTPH1 protein expression and there is a coupling of increased p38gamma and PTPH1 protein expression in primary colon cancer tissues. These results reveal a coordinative oncogenic activity of a MAPK with its specific phosphatase and suggest that PDZ-mediated p38gamma/PTPH1 complex may be a novel target for Ras-dependent malignancies.

p38α Antagonizes p38γ Activity through c-Jun-dependent Ubiquitin-proteasome Pathways in Regulating Ras Transformation and Stress Response

p38 MAPK family consists of four isoform proteins (α, β, γ, and δ) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38α may regulate Ras transformation and stress response through depleting its family member p38γ protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38α (MKK6-p38α) and p38γ (MKK6-p38γ) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38α phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38γ protein expression. p38γ, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38α phosphorylation decreases p38γ protein expression, whereas its inhibition increases cellular p38γ concentrations, indicating an active role of p38α phosphorylation in negatively regulating p38γ protein expression. Mechanistic analyses show that p38α requires c-Jun activation to deplete p38γ proteins by ubiquitin-proteasome pathways. These results suggest that p38α may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38γ family protein.

Baculovirus p35 and Z-VAD-fmk inhibit thapsigargin-induced apoptosis of breast cancer cells

Programmed cell death, or apoptosis, is inhibited by the antiapoptotic oncogene, Bcl-2, and is mediated by a cascade of aspartate-specific cysteine proteases, or caspases, related to interleukin 1-β converting enzyme. Depending on cell type, apoptosis can be induced by treatment with thapsigargin (TG); a selective inhibitor of the endoplasmic reticulum-associated calcium-ATPase. The role of caspases in mediating TG-induced apoptosis was investigated in the Bcl-2-negative human breast cancer cell line, MDA-MB-468. Apoptosis developed in MDA-MB-468 cells over a period of 24 – 72 h following treatment with 100 nM TG, and was prevented by Bcl-2 overexpression. TG-induced apoptosis was associated with activation of caspase-3 and was inhibited by stable expression of the baculovirus p35 protein, an inhibitor of caspase activity. Also, TG-induced apoptosis was inhibited by treating cells with Z-VAD-fmk, a cell-permeable fluoromethylketone inhibitor of caspases. These findings indicate that TG-induced apoptosis of MDA-MB-468 breast cancer cells is subject to inhibition by Bcl-2 and is mediated by caspase activity. This model system should be useful for further investigation directed toward understanding the role of calcium in signaling apoptosis, and its relationship to Bcl-2 and the caspase proteolytic cascade.

p38γ Mitogen-Activated Protein Kinase Integrates Signaling Crosstalk between Ras and Estrogen Receptor to Increase Breast Cancer Invasion

Ras is believed to stimulate invasion and growth by different effector pathways, and yet, the existence of such effectors under physiologic conditions has not been shown. Estrogen receptor (ER), on the other hand, is both anti-invasive and proliferative in human breast cancer, with mechanisms for these paradoxical actions remaining largely unknown. Our previous work showed an essential role of p38gamma mitogen-activated protein kinase in Ras transformation in rat intestinal epithelial cells, and here, we show that p38gamma integrates invasive antagonism between Ras and ER to increase human breast cancer invasion without affecting their proliferative activity. Ras positively regulates p38gamma expression, and p38gamma in turn mediates Ras nonmitogenic signaling to increase invasion. Expression of the Ras/p38gamma axis, however, is trans-suppressed by ER that inhibits invasion and stimulates growth also by distinct mechanisms. Analysis of ER and its cytoplasmic localized mutant reveals that ER additionally binds to p38gamma protein, leading to its specific down-regulation in the nuclear compartment. A p38gamma-antagonistic activity of ER was further shown in a panel of breast cancer cell lines and was shown independent of estrogens by both ER depletion and ER expression. These results revealed that both Ras and ER use distinct pathways to regulate breast cancer growth and invasion, and that p38gamma specifically integrates their antagonistic activity to stimulate cell invasion. Selective targeting of p38gamma-dependent invasion pathways may be a novel strategy to control breast cancer progression.

Stress-induced c-Jun-dependent Vitamin D Receptor (VDR) Activation Dissects the Non-classical VDR Pathway from the Classical VDR Activity

Vitamin D receptor (VDR) is a ligand-dependent transcription factor that mediates vitamin D3-induced gene expression. Our previous work has established that stress MAPK signaling stimulates VDR expression (Qi, X., Pramank, R., Wang, J., Schultz, R. M., Maitra, R. K., Han, J., DeLuca, H. F., and Chen, G. (2002) J. Biol. Chem. 277, 25884–25892) and VDR inhibits cell death in response to p38 MAPK activation (Qi, X., Tang, J., Pramanik, R., Schultz, R. M., Shirasawa, S., Sasazuki, T., Han, J., and Chen, G. (2004) J. Biol. Chem. 279, 22138–22144). Here we show that c-Jun is essential for VDR expression and VDR in turn inhibits c-Jun-dependent cell death by non-classical mechanisms. In response to stress c-Jun is recruited to the Vdr promoter before VDR protein expression is induced. The necessary and sufficient role of c-Jun in VDR expression was established by the fact that c-Jun knock-out decreases VDR expression, whereas c-Jun restoration recovers its activity. Existence of the non-classical VDR pathway was suggested by a requirement of both c-Jun and VDR in stress-induced VDR activity and further demonstrated by VDR inhibiting c-Jun-dependent cell death independent of its classical transcriptional activity and independent of vitamin D3. c-Jun is also required for vitamin D3-induced classical VDR transcriptional activity by a mechanism likely involving physical interactions between c-Jun and VDR proteins. These results together reveal a non-classical mechanism by which VDR acts as a c-Jun/AP-1 target gene to modify c-Jun activity in stress response through increased protein expression independent of classical transcriptional regulations.