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1.
Sci Rep ; 4: 6304, 2014 Sep 09.
Article in English | MEDLINE | ID: mdl-25200404

ABSTRACT

Keratinocyte migration is an early event in the wound healing process. Although we previously found that CD9 downregulation is required for the keratinocyte migration during wound repair, the mechanism of how CD9 expression is regulated remains unclear. Here, we observed the effect of hypoxia (2% O2) on CD9 expression and keratinocyte migration. CD9 expression was downregulated and keratinocyte migration was increased under hypoxic conditions. In addition, CD9 overexpression reversed hypoxia-induced cell migration. We also found that hypoxia activated the p38/MAPK pathway. SB203580, a p38/MAPK inhibitor, increased CD9 expression and inhibited keratinocyte migration under hypoxia, while MKK6 (Glu) overexpression decreased CD9 expression and promoted hypoxic keratinocyte migration. Our results demonstrate that hypoxia regulates CD9 expression and CD9-mediated keratinocyte migration via the p38/MAPK pathway.


Subject(s)
Cell Hypoxia/physiology , Cell Movement/physiology , Tetraspanin 29/biosynthesis , Wound Healing/physiology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Movement/drug effects , Down-Regulation , Enzyme Inhibitors/pharmacology , Humans , Imidazoles/pharmacology , Keratinocytes/metabolism , Keratinocytes/physiology , MAP Kinase Kinase 6/biosynthesis , Mice , Mice, Inbred BALB C , Pyridines/pharmacology , Tetraspanin 29/genetics , Wound Healing/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
2.
Cancer Invest ; 32(8): 416-22, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25019214

ABSTRACT

Expression analysis of MKK6 protein in solid tumors has never been investigated. Here, we report systematic analysis of MKK6 protein in different types of human tumor samples using western blotting and immunofluorescence techniques. We observed significant increase in the expression of MKK6 in Esophageal, Stomach, and Colon cancers as compared to controls. Results were alternately confirmed by Immunofluorescence studies. Upregulation of MKK6 protein is indicative of its role in human cancers and could possibly be used as a novel diagnostic or prognostic marker in these cancers.


Subject(s)
Colonic Neoplasms/enzymology , Esophageal Neoplasms/enzymology , MAP Kinase Kinase 6/biosynthesis , Stomach Neoplasms/enzymology , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Humans , MAP Kinase Kinase 6/genetics , Middle Aged , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Up-Regulation
3.
Protein Pept Lett ; 14(8): 756-60, 2007.
Article in English | MEDLINE | ID: mdl-17979814

ABSTRACT

The conditions were optimized for maximum soluble yield of biologically active recombinant p38alpha mitogen activated protein kinase (MAPK) vis-à-vis insoluble fraction (inclusion body formation). This study reports a rapid, economical and single step purification process for the overproduction of GST tagged p38alpha MAPK. A yield of 18 mg of highly purified and soluble protein per liter of bacterial culture within 6 h timeframe was achieved. The purified protein was found to be biologically suitable for phosphorylation by upstream kinases and was catalytically active. We further demonstrated that our in-house p38alpha MAPK is more potent (>30%) than a commercially available enzyme.


Subject(s)
Escherichia coli/metabolism , Mitogen-Activated Protein Kinase 14/biosynthesis , Cloning, Molecular , Enzyme Activation , Humans , Kinetics , MAP Kinase Kinase 6/biosynthesis , Mitogen-Activated Protein Kinase 14/isolation & purification , Recombinant Proteins/biosynthesis , Solubility
4.
Cancer Sci ; 98(9): 1468-75, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17627613

ABSTRACT

Normal cells irreversibly stop dividing after being exposed to a variety of stresses. This state, called cellular senescence, has recently been demonstrated to act as a tumor-suppressing mechanism in vivo. A common set of features are exhibited by senescent cells, but the molecular mechanism leading to the state is poorly understood. It has been shown that p38, a stress-induced mitogen-activated protein kinase (MAPK), plays a pivotal role in inducing cellular senescence in diverse settings. To better understand the senescence-inducing pathway, microarray analyses of normal human fibroblasts that ectopically activated p38 were performed. It was found that five genes encoding ESE-3, inhibin betaA, RGS5, SSAT and DIO2 were up-regulated in senescent cells induced by RasV12, H(2)O(2) and telomere shortening, but not in quiescent or actively growing cells, suggesting that these genes serve as molecular markers for various types of cellular senescence. The ectopic expression of ESE-3 resulted in retarded growth, up-regulation of p16(INK4a) but not of p21, and increased levels of SA-beta-gal activity. In contrast, RGS5, SSAT and the constitutive active form of the inhibin betaA receptor gene did not induce such senescence phenotypes when ectopically expressed. ESE-3 expression increased the activity of the p16(INK4a) promoter in a reporter assay, and recombinant ESE-3 protein bound to the Ets-binding sequences present in the promoter. These results suggest that ESE-3 plays a role in the induction of cellular senescence as a downstream molecule of p38.


Subject(s)
Cellular Senescence/physiology , Transcription Factors/biosynthesis , Up-Regulation/physiology , Alternative Splicing , Cell Line , Cellular Senescence/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Humans , MAP Kinase Kinase 6/biosynthesis , MAP Kinase Kinase 6/genetics , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/physiology , Trans-Activators/physiology , Transcription Factors/genetics , Transcription Factors/physiology , Up-Regulation/genetics , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/physiology
5.
Cancer Res ; 66(4): 2264-70, 2006 Feb 15.
Article in English | MEDLINE | ID: mdl-16489030

ABSTRACT

Despite considerable efforts to improve early detection of ovarian cancer, the majority of women at time of diagnosis will have metastatic disease. Understanding and targeting the molecular underpinnings of metastasis continues to be the principal challenge in the clinical management of ovarian cancer. Whereas the multistep process of metastasis development has been well established in both clinical and experimental models, the molecular factors and signaling pathways involved in successful colonization of a secondary site by disseminated cancer cells are not well defined. We have previously identified mitogen-activated protein kinase (MAPK) kinase 4/c-Jun NH2-terminal kinase (JNK)-activating kinase (MKK4/JNKK1/SEK1, hereafter referred to as MKK4) as a metastasis suppressor protein in ovarian carcinoma. In this study, we elucidate key mechanisms of MKK4-mediated metastasis suppression. Through the use of a kinase-inactive mutant, we show that MKK4 kinase activity is essential for metastasis suppression and prolongation of animal survival. Because MKK4 can activate either of two MAPKs, p38 or JNK, we expressed MKK6 or MKK7, specific activators of these MAPKs, respectively, to delineate which MAPK signaling module was involved in MKK4-mediated metastasis suppression. We observed that MKK6 expression suppressed metastatic colonization whereas MKK7 had no effect. Our finding that MKK4 and MKK6 both suppress metastasis points to the p38 pathway as an important regulatory pathway for metastatic colonization in ovarian cancer.


Subject(s)
MAP Kinase Kinase 4/metabolism , MAP Kinase Kinase 6/metabolism , MAP Kinase Signaling System/physiology , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/pathology , Animals , Cell Line, Tumor , Female , Humans , MAP Kinase Kinase 4/biosynthesis , MAP Kinase Kinase 6/biosynthesis , MAP Kinase Kinase 7/biosynthesis , MAP Kinase Kinase 7/metabolism , Mice , Mice, SCID , Neoplasm Metastasis , Neoplasm Transplantation , Transplantation, Heterologous
6.
J Biol Chem ; 280(1): 669-76, 2005 Jan 07.
Article in English | MEDLINE | ID: mdl-15492008

ABSTRACT

The mitogen-activated protein kinases (MAPK) have been the subject of many studies to identify signaling pathways that promote cell survival or death. In cultured cardiac myocytes, p38 MAPK promotes cell survival or death depending on whether it is activated by mitogen-activated protein kinase kinase 6 (MKK6) or MKK3, respectively. The objectives of the current study were to examine the effects of MKK6-mediated p38 activation in the heart in vivo. Accordingly, we generated transgenic (TG) mice that overexpress wild type MKK6 in a cardiac-restricted manner. Although p38 was about 17-fold more active in TG than non-transgenic (NTG) mouse hearts, TG mouse hearts were morphologically and functionally similar to those of NTG littermates. However, upon transient ischemia followed by reperfusion, the MKK6 TG mouse hearts exhibited significantly better functional recovery and less injury than NTG mouse hearts. Because MKK6 increases levels of the protective small heat shock protein, alpha B-crystallin (alpha BC), in cultured cardiac myocytes, we examined alpha BC levels in the mouse hearts. The level of alpha BC was 2-fold higher in MKK6 TG than NTG mouse hearts. Moreover, ischemia followed by reperfusion induced a 6.4-fold increase in alpha BC levels in the mitochondrial fractions of TG mouse hearts but no increase in alpha BC levels in any of the other fractions analyzed. These alterations in alpha BC expression and localization suggest possible mechanisms of cardioprotection in MKK6 TG mouse hearts.


Subject(s)
MAP Kinase Kinase 6/biosynthesis , Myocardial Infarction/enzymology , Myocardial Reperfusion Injury/enzymology , Animals , Enzyme Activation , Gene Expression Regulation, Enzymologic , Humans , In Vitro Techniques , MAP Kinase Kinase 3/genetics , MAP Kinase Kinase 3/metabolism , MAP Kinase Kinase 6/genetics , Mice , Mice, Transgenic , Myocardial Infarction/physiopathology , Myocardial Infarction/prevention & control , Myocardial Reperfusion Injury/physiopathology , Organ Specificity , Recovery of Function , alpha-Crystallin B Chain/metabolism
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