Differentiation Of Oral Keratinocytes Research Articles

Intrinsic Differences between the Open Chromatin Regions of Oral and Epidermal Keratinocytes.

To investigate and characterise the differences between the open chromatin regions of oral and epidermal keratinocytes. Human immortalised oral epithelial cell lines (HIOECs) were used as the standard model for oral keratinocytes, and primary normal human epidermal keratinocytes (NHEKs) were chosen as the model for epidermal keratinocytes. Assay for transposase accessible chromatin using sequencing (ATAC-seq) and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) were used to evaluate the dynamic changes in open chromatin regions and active enhancers during oral keratinocyte differentiation. In silico prediction and dual-luciferase assays were used to evaluate the enriched motifs and maintain enhancer activity in specific enriched HIOECs. Integration and comparison of HIOEC ATAC-seq with NHEK ATAC-seq were used to identify oral keratinocyte-enriched open chromatin regions along with key motifs governing differential enhancer activity. The genomic regulatory elements and GWAS overlap algorithm was used to compare the annotation rate of HIOEC-overlapped craniofacial enhancers with other craniofacial enhancers for orofacial cleft-associated variants. During the differentiation of HIOECs, 14933 open chromatin regions became more accessible. Grainyhead-like (GRHL) and Krüppel-like factor (KLF) motifs were overrepresented in maintaining HIOEC-specific activity. Compared with NHEKs, 16161 open chromatin regions were uniquely accessible in HIOECs. Within these regions, the C/EBP motif governed HIOEC-specific enhancer regulating SOX2 and PITX2, which enhanced oral keratinocyte wound healing. When intersected with human craniofacial super-enhancers, open chromatin regions in HIOECS can better annotate the common variants associated with orofacial cleft. The intrinsic differences between the open chromatin regions of human oral and epidermal keratinocytes are directly maintained by a set of transcription factors.

The lytic activation of KSHV during keratinocyte differentiation is dependent upon a suprabasal position, the loss of integrin engagement, and calcium, but not the interaction of cadherins

We previously found that KSHV (HHV-8) lytic activation occurs during differentiation of oral keratinocytes in organotypic raft cultures. To further investigate the spatial and temporal aspects of KSHV lytic activation and the roles of integrins, cadherins, and calcium, we used rKSHV.219-infected primary oral keratinocytes in submerged, suspension, and direct suprabasal plating, models of differentiation. We found that early keratinocyte differentiation did not activate lytic KSHV in cells attached to a substratum, with activation only occurring in suprabasal cells. Temporally, KSHV lytic expression occurred between the expression of early and late differentiation markers. Keratinocytes differentiated in suspension culture, which mimics substratum loss that occurs with stratification, activated lytic KSHV. This lytic activation was inhibited by integrin engagement, showing that integrins are a control point for KSHV reactivation. A role for cadherins was not found. Elevated extracellular calcium was necessary, but not sufficient, for lytic activation.

Expression of basal cell marker revealed by RAM11 antibody during epithelial regeneration in rabbits.

RAM11 is a mouse monoclonal anti-rabbit macrophage antibody recognizing connective tissue and vascular macrophages. Our previous report showed that RAM11 reacted with basal cells of stratified squamous epithelia of rabbit skin, oral mucosa and esophagus. The aim of the present study was to follow the appearance of RAM11 immunoreactivity in basal cells of regenerating oral epithelium in rabbits. No RAM11 immunostaining was observed in the regenerating epithelium examined on days 1 and 3 of wound healing. A weak immunofluorescence first appeared on day 7 in single basal cells and 32% of RAM11- positive basal cells were observed on day 14. These findings indicate that expression of the antigen recognized by RAM11 antibody is a transient event in the differentiation of oral keratinocytes which not always occurs during epithelial repair, although it is a constant feature of epithelial turnover in mature epithelium. Therefore this antigen can be regarded as basal cell marker only in mature stratified squamous epithelia.

Snail Up-regulates Proinflammatory Mediators and Inhibits Differentiation in Oral Keratinocytes

The transcriptional repressor Snail2 is overexpressed in head and neck squamous cell carcinomas (HNSCC) relative to nonmalignant head and neck mucosal epithelium, and in locally recurrent relative to nonrecurrent HNSCCs. We investigated the mechanisms by which Snails might contribute to the pathogenesis of HNSCCs using cell biological and molecular analyses. Oral keratinocytes that expressed Snails acquired an enhanced ability to attract monocytes and to invade a dense interstitial collagen matrix. They were also found to up-regulate production of proinflammatory cytokines and cyclooxygenase-2 (COX2), which have previously been shown to correlate with malignancy. Induction of nuclear factor-kappaB transcriptional activity by Snails was weak and not sufficient to account for the elevated levels of COX2, interleukin (IL)-6, IL8, or CXCL1. In addition, expression of Snails in oral keratinocytes impaired desquamation in vitro and strongly repressed expression of both ELF3 and matriptase-1, which play important roles in the terminal differentiation of keratinocytes. Reexpression of matriptase-1 in Snail-expressing cells partially rescued desquamation. This implicates Snails as contributing to malignancy both at the early stages, by impeding terminal differentiation, and at later stages, when invasion and inflammation are important.

Regulation of IGFBP-5 expression during tumourigenesis and differentiation of oral keratinocytes.

To identify molecular events involved in the pathogenesis of oral squamous cell carcinoma (OSCC), genes differentially expressed in OSCC and non-cancerous matched tissue (NCMT) samples were analysed using a subtractive hybridization strategy. NCMT-enriching clones that have been linked to suppressor pathway in previous studies were subjected to advanced analyses. Complete absence of insulin-like growth factor binding protein-5 (IGFBP-5) expression at both the mRNA and the protein level was identified in nearly all (5/6) OSCC cell lines with the exception of the SCC25 cell line, which exhibited high IGFBP-5 expression. However, this protein is consistently present in cultured normal human oral keratinocytes (NHOKs). Immunohistochemistry revealed moderate to strong cytoplasmic immunoreactivity of IGFBP-5 in the stratum spinosum and stratum granulosum in the vast majority of NCMT samples. A remarkable reduction in IGFBP-5 immunoreactivity was detected in 56% (26/46) of OSCC samples, compared with the corresponding NCMT (p < 0.0001). Induction of differentiation in both NHOKs and SCC25 up-regulated IGFBP-5 expression. Administration of a green tea compound with anti-cancer properties, (-)-epigallocatechin 3-gallate, at a concentration of 5-20 micro g/ml also up-regulated IGFBP-5 expression in NHOKs in a dose-dependent manner. The findings suggest that IGFBP-5 may be an important factor in the differentiation of oral keratinocytes and that down-regulation of IGFBP-5 may be involved in the neoplastic transformation of oral keratinocytes.

Retinoic Acid Regulates Oral Epithelial Differentiation by Two Mechanisms

The effect of retinoic acid (RA) concentration on differentiation of oral keratinocytes and the influence of fibroblasts on RA-dependent regulation were investigated in a lifted culture system. Keratinocyte differentiation was assessed by morphology, immunohistochemistry and immunoblotting. Filaggrin/profilaggrin and keratin 1 were used as biochemical markers for cornified epithelium and keratins 13 and 19 as markers for noncornified epithelium. Cultured oral keratinocytes in RA-free conditions differentiated in a manner that closely resembled the differentiation pattern of gingival epithelia in vivo. Increasing RA concentrations altered the in vivo-like terminal differentiation of oral keratinocytes by disruption of organized stratification, inhibition of filaggrin/profilaggrin and K1 expression, and stimulation of K13 and K19 expression. Differentiation of keratinocytes from both cornified and noncornified regions of the oral cavity varied in a similar manner in response to added RA, with the exception of K19 expression. K19 was consistently expressed at higher levels in keratinocytes originating from noncornified epithelial as compared to those from cornified epithelia. The level of RA regulation was ultimately dependent on the type of fibroblasts underlying the epithelial cells. Homologous fibroblasts rendered the oral keratinocytes less sensitive to the effects of RA than skin fibroblasts. In addition, at a given RA concentration, fibroblasts from cornified oral mucosa potentiated keratinocyte expression of RA sensitive markers of keratinization as compared to the influence exerted by fibroblasts originating from noncornified oral mucosa. These results indicate that the RA regulation of oral epithelial differentiation is mediated by two separate mechanisms: a direct, RA concentration-dependent effect, and an indirect, fibroblast-mediated effect.