A Limited Role for Retinoic Acid and Retinoic Acid Receptors RARα and RARβ in Regulating Keratin 19 Expression and Keratinization in Oral and Epidermal Keratinocytes

Different types of stratified squamous epithelia—for example, the “orthokeratinized” epidermis, the “parakeratinized” gingiva, and the “nonkeratinized” oral lining mucosal epithelia—are formed by intrinsically distinct keratinocyte subtypes. These subtypes exhibit characteristic patterns of keratin protein expression in vivo and in culture. Keratin 19 is an informative subtype-specific marker because the basal cells of only nonkeratinizing epithelia express K19 in vivo and in culture. Epidermal keratinocytes normally do not express K19, but can be induced to do so in culture by retinoic acid (RA). Keratinocyte subtypes express the retinoic acid receptor (RAR)β at levels roughly correlated with their level of K19 expression in culture and their potential for forming a nonkeratinized epithelium in vivo. We tested the hypothesis that the level of RARβ expressed by a keratinocyte determines its K19 expression and its form of suprabasal differentiation. Normal human epidermal and gingival keratinocytes stably overexpressing either RARβ or RARα were generated by defective retroviral transduction. Overexpression of either receptor enhanced the RA inducibility of K19 in conventional culture, in that the proportion of the transductants becoming K19+ in response to RA was markedly increased compared with controls. The pattern of differentiation of the epithelium formed in organotypic culture, assessed by basal K19 and suprabasal K1, K4, and filaggrin expression, however, was unaltered by PAR overexpression. Thus, the susceptibility of keratinocytes to regulation of K19 expression by retinoids is conditional, and levels of neither RARβ nor RARα are limiting to the intrinsic mechanism that specifies alternate differentiation pathways for stratified squamous epithelia

RSK Activation of Translation Factor eIF4B Drives Abnormal Increases of Laminin γ2 and MYC Protein during Neoplastic Progression to Squamous Cell Carcinoma

Overexpression of the basement membrane protein Laminin γ2 (Lamγ2) is a feature of many epidermal and oral dysplasias and all invasive squamous cell carcinomas (SCCs). This abnormality has potential value as an immunohistochemical biomarker of premalignancy but its mechanism has remained unknown. We recently reported that Lamγ2 overexpression in culture is the result of deregulated translation controls and depends on the MAPK-RSK signaling cascade. Here we identify eIF4B as the RSK downstream effector responsible for elevated Lamγ2 as well as MYC protein in neoplastic epithelial cells. Premalignant dysplastic keratinocytes, SCC cells, and keratinocytes expressing the E6 oncoprotein of human papillomavirus (HPV) type 16 displayed MAPK-RSK and mTOR-S6K1 activation and overexpressed Lamγ2 and MYC in culture. Immunohistochemical staining of oral dysplasias and SCCs for distinct, RSK- and S6K1-specific S6 phosphorylation events revealed that their respective upstream pathways become hyperactive at the same time during neoplastic progression. However, pharmacologic kinase inhibitor studies in culture revealed that Lamγ2 and MYC overexpression depends on MAPK-RSK activity, independent of PI3K-mTOR-S6K1. eIF4B knockdown reduced Lamγ2 and MYC protein expression, consistent with the known requirement for eIF4B to translate mRNAs with long, complex 5′ untranslated regions (5′-UTRs). Accordingly, expression of a luciferase reporter construct preceded by the Lamγ2 5′-UTR proved to be RSK-dependent and mTOR-independent. These results demonstrate that RSK activation of eIF4B is causally linked to elevated Lamγ2 and MYC protein levels during neoplastic progression to invasive SCC. These findings have potential clinical significance for identifying premalignant lesions and for developing targeted drugs to treat SCC

Expression of RAS oncogene in cultured human cells alters the transcriptional and post transcriptional regulation of cytokine genes

Autonomous production of cytokines such as the hematopoietic colony-stimulating factors (CSFs), IL-1, or IL-6 has been demonstrated in numerous human and murine neoplasms, and may be involved in the pathogenesis of several paraneoplastic syndromes such as leukocytosis, fever, and hypercalcemia. Because of the high frequency with which mutations in ras protooncogenes have been detected in human tumors, as well as evidence linking ras gene products to activation of certain cellular functions, we investigated whether ras mutations might influence the regulation of cytokine genes. Normal human fibroblasts transfected with a mutant val"2 H-ras oncogene expressed increased levels of mRNA transcripts encoding granulocyte-CSF (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and IL-1, compared with controls. Human mesothelioma cells transfected with a mutant asp'2 N-ras oncogene exhibited similar alterations in cytokine gene expression. Estimates of transcriptional activity by nuclear run-on analysis revealed a selective increase in transcription only for the IL-1 gene. Analysis of mRNA half-life demonstrated a marked increase in the stability of numerous cytokine transcripts, including G-CSF, GM-CSF, IL-1, and IL-6. The addition of anti-IL-I neutralizing antibody to cultures of cells expressing ras mutants did not block the expression of any of the cytokines examined, suggesting that the baseline expression of GM-CSF, G-CSF, and IL-6 was not a secondary event due to the increased transcription of IL-1. These results indicate that mutations in ras genes may alter expression of several cytokine genes through both transcriptional and posttranscriptiona

MAPK/RSK-dependent, mTOR/S6K1-independent activation of eIF4B and Lamγ2 mRNA translation.

<p><b>A</b>) Western blot analysis of confluent cultures of SCC-68, the premalignant oral keratinocyte line POE9n, and normal primary keratinocyte strain N engineered to express the JH26 mutant of HPV16 E6 (N/E6(JH26). Cultures were treated for 24 hr with the indicated kinase inhibitors and then analyzed for levels of Lamγ2 and MYC protein and for the phosphorylated, activated forms of signaling proteins and translation factors. The Lamγ2 band shown is the 155 kD intracellular form and not the 105 kD form that predominates after secretion and proteolytic processing. The bar graphs below show densitometric analysis of Lamγ2 and p-EIF4B levels in each drug treatment condition relative to untreated control cultures of each line, as described in panel B. <b>B</b>) SCC-13 cells transfected with the reporter constructs pDL-N, pDL-N/(Lamγ2 5′-UTR), and pDL-N/(ODC 5-′UTR) with or without the RSK inhibitor BI-D1870 or the mTORC1/2 inhibitor Ku-0063794 and analyzed for Renilla and Firefly luciferase activity. Reduction caused by BI-D1780 in Lamγ2 5′UTR- and ODC 5′UTR-dependent expression had P values for significance of 0.0043 and 0.01, respectively.</p

Human oral epithelial lesions examined for increased p-S6 and Laminin γ2 by immunohistochemical staining.

<p>Formalin-fixed, paraffin-embedded specimens of oral lesions were immunostained for p-S6(S235), p-S6(S240), and Lamγ2 and the percentage of the total dysplasia positive in the basal cell layer for these markers determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078979#s2" target="_blank">Methods</a>. Basal layer p-S6(235) and p-S6(240) immunostaining always corresponded precisely. n.d.: not determined.</p

Coincidence of p-S6(S235) and p-S6(S240) detectable in human oral dysplastic lesions and SCCs <i>in vivo</i>.

<p>Sections of normal (<b>A</b>) and dysplastic (<b>B-C</b>) epithelium, and invasive SCC (<b>D</b>), stained with H&E and immunostained for Lamγ2, p-S6(S240) and p-S6(S235). Scale bar: 200 µm. Enlarged insets of some regions are shown for easier viewing of p-S6 staining patterns. Panel A shows a region of Case 2, panel B of case 8, panel C of case 7, and panel D of case 4 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078979#pone-0078979-t001" target="_blank">Table 1</a>. Note that normal epithelium did not express Lamγ2 and neither S6 phosphorylation event was detectable in the basal cells. Cells in dysplasias and SCCs always showed coincidence of the two S6 phosphorylation events. Dysplasias varied with respect to frequency and intensity of Lamγ2 expression and S6 phosphorylation, with Lamγ2 cells representing a subset of p-S6 positive basal cells and invasive SCCs contained many Lamγ2 and basal layer p-S6 positive regions.</p