Homeobox transcription factors play important roles in epidermal renewal. Among them HOXA5 emerges as a promising member. However, its direct effect on epidermal biology, either to promote or to inhibit growth, is still controversial.
We proposed to unravel the role of HOXA5 in modulating keratinocytes growth and epidermal formation in organotypic cultures both in vitro and in vivo.
We transfected HaCaT cells with lentivirual vectors which over-expressed either wild-type or mutant HOXA5 cDNAs with deleted homeodomain. Subsequently we propagated the cells in organotypic cultures (OTCs) and then transplanted them into nude mice. Cell proliferation and cell cycle progression were detected. Epidermal morphogenesis and stratification were investigated by immunohistochemistry and immunofluorescence staining of a series of epidermal markers.
HaCaT cells transfected with HOXA5 cDNAs displayed lower growth rate and delayed G1-S transition. HOXA5-transfected OTC exhibited an aberrantly organized epithelium with significantly increased TUNEL staining as well as decreased PCNA and K5 staining, while expression of differentiation markers as K10, involucrin and filaggrin were somewhat enhanced. However, under in vivo environment in nude mice which had great paracrine regulatory mechanisms, the aberrant phenotype was ameliorated as shown by a more regular tissue organization and normal expression of PCNA and K5. Inversely, cells transfected with the homeodomain-deleted protein exhibited accelerated growth and produced a more proliferative and better-orchestrated epidermis, as shown by well-expressed proliferation and differentiation markers.
HOXA5 can suppress keratinocytes growth and epidermal formation. It probably activated antagonist genes against growth factors release, which depends on its homeodomain.
[Show abstract][Hide abstract] ABSTRACT: RanBPM is a ubiquitous protein that has been reported to regulate several cellular processes through interactions with various proteins. However, it is not known whether RanBPM may regulate gene expression patterns. As it has been shown that RanBPM interacts with a number of transcription factors, we hypothesized that it may have wide ranging effects on gene expression that may explain its function. To test this hypothesis, we generated stable RanBPM shRNA cell lines to analyze the effect of RanBPM on global gene expression. Microarray analyses were conducted comparing the gene expression profile of Hela and HCT116 RanBPM shRNA cells versus control shRNA cells. We identified 167 annotated genes significantly up- or down-regulated in the two cell lines. Analysis of the gene set revealed that down-regulation of RanBPM led to gene expression changes that affect regulation of cell, tissue, and organ development and morphology, as well as biological processes implicated in tumorigenesis. Analysis of Transcription Factor Binding Sites (TFBS) present in the gene set identified several significantly over-represented transcription factors of the Forkhead, HMG, and Homeodomain families of transcription factors, which have previously been demonstrated as having important roles in development and tumorigenesis. In addition, the combined results of these analyses suggested that several signaling pathways were affected by RanBPM down-regulation, including ERK1/2, Wnt, Notch, and PI3K/Akt pathways. Lastly, analysis of selected target genes by quantitative RT-qPCR confirmed the changes revealed by microarray. Several of the genes up-regulated in RanBPM shRNA cells encode proteins with known oncogenic functions, such as the RON tyrosine kinase, the adhesion molecule L1CAM, and transcription factor ELF3/ESE-1, suggesting that RanBPM functions as a tumor suppressor to prevent deregulated expression of these genes. Altogether, these results suggest that RanBPM does indeed function to regulate many genomic events that regulate embryonic, tissue, and cellular development as well as those involved in cancer development and progression.
American Journal of Cancer Research 09/2012; 2(5):549-65. · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNA-Seq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes.
Experimental Cell Research 09/2013; 320(1). DOI:10.1016/j.yexcr.2013.09.011 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HoxA5 is expressed in quiescent endothelial cells (EC), but absent in activated angiogenic EC. To examine the efficacy of targeting HoxA5 therapeutically to quell pathologic or tumor angiogenesis, we generated an inducible, transgenic mouse model of sustained HoxA5 expression in ECs. During pathologic angiogenesis, sustained HoxA5 regulates expression several angiogenic effector molecules, notably increased expression of TSP-2 and reduced expression of VEGF, thus leading to inhibition of pathological angiogenesis in tissues. To evaluate if this impressive reduction of vascularization could also impact tumor angiogenesis, HoxA5 mice were bred with a mouse model of de novo squamous carcinogenesis, e.g., K14-HPV16 mice. Activation of EC-HoxA5 significantly reduced infiltration by mast cells into neoplastic skin, an early hallmark of progression to dysplasia, reduced angiogenic vasculature, and blunted characteristics of tumor progression. To evaluate HoxA5 as a therapeutic, topical application of a HoxA5 transgene onto early neoplastic skin of K14-HPV16 mice similarly resulted in a significant impairment of angiogenic vasculature and progression to dysplasia to a similar extent as observed with genetic delivery of HoxA5. Together these data indicate that HoxA5 represents a novel molecule for restricting pathological and tumorigenic angiogenesis.
PLoS ONE 03/2015; 10(3):e0121720. DOI:10.1371/journal.pone.0121720 · 3.23 Impact Factor
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