ABSTRACT
Fatty acid-binding proteins (FABPs) are intracellular lipid-binding proteins that play roles in fatty acid transport and the regulation of gene expression. Dysregulated FABP expression and/or activity have been associated with cancer pathogenesis; in particular, epidermal-type FABP (FABP5) is upregulated in many types of cancer. However, the mechanisms regulating FABP5 expression and its involvement in cancer remain largely unknown. Here, we examined the regulation of FABP5 gene expression in non-metastatic and metastatic human colorectal cancer (CRC) cells. We found that FABP5 expression was upregulated in metastatic compared with non-metastatic CRC cells as well as in human CRC tissues compared with adjacent normal tissue. Analysis of the DNA methylation status of the FABP5 promoter showed that hypomethylation correlated with the malignant potential of the CRC cell lines. Moreover, FABP5 promoter hypomethylation also correlated with the expression pattern of splice variants of the DNA methyltransferase DNMT3B. ChIP assays and luciferase reporter assays demonstrated that the transcription factor nuclear factor-kappa B (NF-κB) was involved in regulating FABP5 expression. FABP5 expression could be upregulated in metastatic CRC cells by sequential promotion of DNA demethylation followed by activation of NF-κB. We also found that upregulated FABP5 in turn controlled NF-κB activity through IL-8 production. Collectively, these findings suggest the existence of a DNA methylation-dependent NF-κB /FABP5 positive feed-forward loop that may lead to constitutive activation of NF-κB signaling pathway and play a crucial role in CRC progression.
Subject(s)
Colonic Neoplasms , NF-kappa B , Humans , NF-kappa B/metabolism , DNA Methylation , Signal Transduction , Colonic Neoplasms/genetics , Fatty Acid-Binding Proteins/genetics , Fatty Acid-Binding Proteins/metabolismABSTRACT
In mammals, γ-tubulin, a key protein in microtubule nucleation, is encoded by two genes, TUBG1 and TUBG2. Human TUBG1 and TUBG2 mRNA are expressed ubiquitously and predominantly in preimplantation embryos and the brain, respectively, but specific detection of γ-tubulin2 protein expression is difficult due to their high sequence similarity. Here, we describe a protocol for differential detection of two human γ-tubulins by western blotting. In several cancer cell lines and the brain, expression of γ-tubulin2 along with γ-tubulin1 and a novel TUBG2 splice variant are identified. Contribution of ectopically expressed γ-tubulin2 in cancer growth was determined by depletion of γ-tubulin2.
Subject(s)
Gene Expression Regulation , RNA Splicing/genetics , Tubulin/genetics , Base Sequence , Brain/metabolism , Cell Line , Cell Proliferation , Down-Regulation/genetics , Humans , RNA, Small Interfering/metabolism , Transfection , Tubulin/metabolismABSTRACT
The phenotypes of mice lacking peptidyl prolyl cis/trans isomerase Pin1 (Pin1(-/-)) indicated that deficient Pin1 might be related to a variety of diseases. We created TAT-Pin1, a fusion protein of human immunodeficiency virus 1 trans-activator of transcription factor with Pin1. Treatment of HeLa cells with TAT-Pin1 increased the ratio of the S phase. Moreover, TAT-Pin1 restored the proliferating function of Pin1(-/-) mouse embryonic fibroblasts which cannot restart proliferation after G0 arrest. These results indicate that TAT-Pin1 is useful in studying the functions of Pin1 and can be developed as a macromolecular drug for diseases related to Pin1 loss.
Subject(s)
Peptidylprolyl Isomerase/chemistry , Peptidylprolyl Isomerase/metabolism , Protein Engineering/methods , Amino Acid Sequence , Animals , HeLa Cells , Humans , Mice , Models, Molecular , NIMA-Interacting Peptidylprolyl Isomerase , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptidylprolyl Isomerase/genetics , Peptidylprolyl Isomerase/pharmacology , Protein Transport , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , S Phase/drug effects , tat Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
Here, we report a novel role for hGas7b (human growth arrest specific protein 7b) in the regulation of microtubules. Using a bioinformatic approach, we studied the actin-binding protein hGas7b with a structural similarity to the WW domain of a peptidyl prolyl cis/trans isomerase, Pin1, that facilitates microtubule assembly. Thus, we have demonstrated that hGas7b binds Tau at the WW motif and that the hGas7b/Tau protein complex interacts with the microtubules, promoting tubulin polymerization. Tau, in turn, contributes to protein stability of hGas7b. Furthermore, we observed decreased levels of hGas7b in the brains from patients with Alzheimer disease. These results suggest an important role for hGas7b in microtubular maintenance and possible implication in Alzheimer disease.
