RESUMEN
Models of skin diseases, such as psoriasis and scleroderma, must accurately recapitulate the complex microenvironment of human skin to provide an efficacious platform for investigation of skin diseases. Skin disease research has been shifting from less complex and less relevant 2D (two-dimensional) models to significantly more relevant 3D (three-dimensional) models. Three-dimensional modeling systems are better able to recapitulate the complex cell-cell and cell-matrix interactions that occur in vivo within skin. Three-dimensional human skin equivalents (HSEs) have emerged as an advantageous tool for the study of skin disease in vitro. These 3D HSEs can be highly complex, containing both epidermal and dermal compartments with integrated adnexal structures. The addition of adnexal structures to 3D HSEs has allowed researchers to gain more insight into the complex pathology of various hereditary and acquired skin diseases. One method of constructing 3D HSEs, 3D bioprinting, has emerged as a versatile and useful tool for generating highly complex HSEs. The development of commercially available 3D bioprinters has allowed researchers to create highly reproducible 3D HSEs with precise integration of multiple adnexal structures. While the field of bioengineered models for study of skin disease has made tremendous progress in the last decade, there are still significant efforts necessary to create truly biomimetic skin disease models. In future studies utilizing 3D HSEs, emphasis must be placed on integrating all adnexal structures relevant to the skin disease under investigation. Thorough investigation of the intricate pathology of skin diseases and the development of effective treatments requires use of highly efficacious models of skin diseases.
RESUMEN
Pactamycin, although putatively touted as a potent antitumor agent, has never been used as an anticancer drug due to its high cytotoxicity. In this study, we characterized the effects of two novel biosynthetically engineered analogs of pactamycin, de-6MSA-7-demethyl-7-deoxypactamycin (TM-025) and 7-demethyl-7-deoxypactamycin (TM-026), in head and neck squamous cell carcinoma (HNSCC) cell lines SCC25 and SCC104. Both TM-025 and TM-026 exert growth inhibitory effects on HNSCC cells by inhibiting cell proliferation. Interestingly, unlike their parent compound pactamycin, the analogs do not inhibit synthesis of nascent protein in a cell-based assay. Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines. Cell cycle analysis demonstrated that both analogs significantly induce cell cycle arrest of the HNSCC cells at S-phase resulting in reduced accumulation of G2/M-phase cells. The pactamycin analogs induce expression of cell cycle regulatory proteins including master regulator p53, its downstream target p21Cip1/WAF1, p27kip21, p19, cyclin E, total and phospho Cdc2 (Tyr15) and Cdc25C. Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4. Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase. Altogether, our results demonstrate that Pactamycin analogs TM-025 and TM-026 induce senescence and inhibit proliferation of HNSCC cells via accumulation in S-phase through possible contribution of p53. The two PCT analogs can be widely used as research tools for cell cycle inhibition studies in proliferating cancer cells with specific mechanisms of action.
Asunto(s)
Carcinoma de Células Escamosas/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Neoplasias de Cabeza y Cuello/patología , Hidrocarburos Fluorados/farmacología , Pactamicina/análogos & derivados , Fase S/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Silenciador del Gen/efectos de los fármacos , Humanos , Modelos Biológicos , Pactamicina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Carcinoma de Células Escamosas de Cabeza y Cuello , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: COUP-TF interacting protein 2 [(Ctip2), also known as Bcl11b] is an important regulator of skin homeostasis, and is overexpressed in head and neck cancer. Ctip2(ep-/-) mice, selectively ablated for Ctip2 in epidermal keratinocytes, exhibited impaired terminal differentiation and delayed epidermal permeability barrier (EPB) establishment during development, similar to what was observed in Ctip2 null (Ctip2(-/-)) mice. Considering that as an important role of Ctip2, and the fact that molecular networks which underlie cancer progression partially overlap with those responsible for tissue remodeling, we sought to determine the role of Ctip2 during cutaneous wound healing. METHODOLOGY/PRINCIPAL FINDINGS: Full thickness excisional wound healing experiments were performed on Ctip2(L2/L2) and Ctip2(ep-/-) animals per time point and used for harvesting samples for histology, immunohistochemistry (IHC) and immunoblotting. Results demonstrated inherent defects in proliferation and migration of Ctip2 lacking keratinocytes during re-epithelialization. Mutant mice exhibited reduced epidermal proliferation, delayed keratinocyte activation, altered cell-cell adhesion and impaired ECM development. Post wounding, Ctip2(ep-/-) mice wounds displayed lack of E-Cadherin suppression in the migratory tongue, insufficient expression of alpha smooth muscle actin (alpha SMA) in the dermis, and robust induction of K8. Importantly, dysregulated expression of several hair follicle (HF) stem cell markers such as K15, NFATc1, CD133, CD34 and Lrig1 was observed in mutant skin during wound repair. CONCLUSIONS/SIGNIFICANCE: Results confirm a cell autonomous role of keratinocytic Ctip2 to modulate cell migration, proliferation and/or differentiation, and to maintain HF stem cells during cutaneous wounding. Furthermore, Ctip2 in a non-cell autonomous manner regulated granulation tissue formation and tissue contraction during wound closure.
Asunto(s)
Regulación de la Expresión Génica , Folículo Piloso/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Piel/patología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Actinas/biosíntesis , Animales , Animales Recién Nacidos , Cadherinas/biosíntesis , Diferenciación Celular , Movimiento Celular , Progresión de la Enfermedad , Inmunohistoquímica/métodos , Queratinocitos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Faloidina/biosíntesis , Piel/metabolismo , Células Madre/citología , Tretinoina/metabolismo , Cicatrización de HeridasRESUMEN
A defective skin epidermal permeability barrier (EPB) is responsible for a high mortality rate in premature infants and is an important risk factor in inflammatory skin diseases such as eczema. We report here fast and accurate methods for measurement of EPB in animal models or in human patients using simple techniques that monitor diffusion of dyes (X-Gal or Lucifer Yellow) through the upper epidermis and measure transepidermal water loss (TEWL) resulting from a defective skin barrier. Accurate diagnosis and early detection of EPB defects in human patients are critical for effective treatment of certain classes of inflammatory skin diseases.
Asunto(s)
Embrión de Mamíferos/metabolismo , Epidermis/metabolismo , Colorantes Fluorescentes/análisis , Galactósidos/análisis , Indoles/análisis , Isoquinolinas/análisis , Queratinocitos/metabolismo , Proteínas Represoras/deficiencia , Proteínas Supresoras de Tumor/deficiencia , Animales , Diferenciación Celular , Difusión , Eccema/metabolismo , Eccema/patología , Células Epidérmicas , Epidermis/embriología , Femenino , Colorantes Fluorescentes/metabolismo , Galactósidos/metabolismo , Humanos , Técnicas In Vitro , Indoles/metabolismo , Lactante , Isoquinolinas/metabolismo , Queratinocitos/citología , Mamíferos , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Microscopía Fluorescente , Permeabilidad , Embarazo , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genética , Pérdida Insensible de AguaRESUMEN
Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. In the absence of keratinocytic RXRα, in combination with mutant Cdk4, cutaneous melanoma was generated that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgfâ2â) was elevated in skin of bigenic mice, whereas Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/metabolismo , Células Epidérmicas , Queratinocitos/fisiología , Melanoma , Comunicación Paracrina , Receptor alfa X Retinoide/metabolismo , Neoplasias Cutáneas , Animales , Quinasa 4 Dependiente de la Ciclina/genética , Progresión de la Enfermedad , Endotelina-1/genética , Endotelina-1/metabolismo , Regulación de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Queratinocitos/citología , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Receptor alfa X Retinoide/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
The TAF4 subunit of transcription factor TFIID was inactivated in the basal keratinocytes of foetal and adult mouse epidermis. Loss of TAF4 in the foetal epidermis results in reduced expression of the genes required for skin barrier function, leading to early neonatal death. By contrast, TAF4 inactivation in adult epidermis leads to extensive fur loss and an aberrant hair cycle characterised by a defective anagen phase. Although the mutant epidermis contains few normal anagen-phase hair follicles, many genes expressed at this stage are strongly upregulated indicating desynchronized and inappropriate gene expression. The TAF4 mutant adult epidermis also displays interfollicular hyperplasia associated with a potent upregulation of several members of the EGF family of mitogens. Moreover, loss of TAF4 leads to malignant transformation of chemically induced papillomas and the appearance of invasive melanocytic tumours. Together, our results show that TAF4 is an important regulator of keratinocyte proliferation and has cell-autonomous and non-cell-autonomous tumour suppressor activity.
Asunto(s)
Proliferación Celular , Epidermis/metabolismo , Queratinocitos/citología , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factores Asociados con la Proteína de Unión a TATA/fisiología , Factor de Transcripción TFIID/metabolismo , Factor de Transcripción TFIID/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Diferenciación Celular/genética , Epidermis/embriología , Epidermis/patología , Femenino , Predisposición Genética a la Enfermedad , Cabello/citología , Cabello/embriología , Hiperplasia/inducido químicamente , Masculino , Ratones , Ratones Noqueados , Nevo Pigmentado/inducido químicamente , Nevo Pigmentado/genética , Subunidades de Proteína/fisiología , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/patología , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Tretinoina/efectos adversos , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
COUP-TF-interacting protein 2 (CTIP2), also known as Bcl11b, is a transcriptional regulatory protein that is highly expressed in and plays a critical role(s) during development of T lymphocytes and the central nervous system. We demonstrate herein that CTIP2 is also highly expressed in mouse skin during embryogenesis and in adulthood as revealed by immunohistochemical analyses. CTIP2 expression in the ectoderm was first detected at embryonic day 10.5 (E10.5), and became increasingly restricted to proliferating cells of the basal cell layer of the developing epidermis in later stages of fetal development and in adult skin. In addition, CTIP2 expression was also detected in some cells of the suprabasal layer of the developing epidermis, as well as in developing and mature hair follicles. Relatively fewer cells of the developing dermal component of skin were found to express CTIP2, and the adult dermis was devoid of CTIP2 expression. Some, but not all, of the cells present within hair follicle bulge were found to co-express CTIP2, keratin K15, but not CD34, indicating that a subset of K15(+) CD34(-) skin stem cells may express CTIP2. Considered together, these findings suggest that CTIP2 may play a role(s) in skin development and/or homeostasis.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica , Proteínas Represoras/biosíntesis , Piel/embriología , Proteínas Supresoras de Tumor/biosíntesis , Animales , Antígenos CD34/biosíntesis , Proliferación Celular , Epidermis/metabolismo , Inmunohistoquímica , Queratina-15/biosíntesis , Ratones , Ratones Endogámicos ICR , Linfocitos T/metabolismo , Factores de TiempoRESUMEN
Retinoid-X-receptor alpha (RXRalpha), a member of the nuclear receptor (NR) superfamily, is a ligand-dependent transcriptional regulatory factor. It plays a crucial role in NR signalling through heterodimerization with some 15 NRs. We investigated the role of RXRalpha and its partners on mouse skin tumor formation and malignant progression upon topical DMBA/TPA treatment. In mutants selectively ablated for RXRalpha in keratinocytes, epidermal tumors increased in size and number, and frequently progressed to carcinomas. As keratinocyte-selective peroxisome proliferator-activated receptor gamma (PPARgamma) ablation had similar effects, RXRalpha/PPARgamma heterodimers most probably mediate epidermal tumor suppression. Keratinocyte-selective RXRalpha-null and vitamin-D-receptor null mice also exhibited more numerous dermal melanocytic growths (nevi) than control mice, but only nevi from RXRalpha mutant mice progressed to invasive human-melanoma-like tumors. Distinct RXRalpha-mediated molecular events appear therefore to be involved, in keratinocytes, in cell-autonomous suppression of epidermal tumorigenesis and malignant progression, and in non-cell-autonomous suppression of nevi formation and progression. Our study emphasizes the crucial role of keratinocytes in chemically induced epidermal and melanocytic tumorigenesis, and raises the possibility that they could play a similar role in UV-induced tumorigenesis, notably in nevi formation and progression to melanoma.
Asunto(s)
Transformación Celular Neoplásica/patología , Epidermis/metabolismo , Queratinocitos/metabolismo , Nevo Pigmentado/patología , Papiloma/patología , Receptor alfa X Retinoide/metabolismo , Neoplasias Cutáneas/patología , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinógenos , Transformación Celular Neoplásica/inducido químicamente , Epidermis/patología , Regulación Neoplásica de la Expresión Génica , Queratinocitos/patología , Ratones , Ratones Transgénicos , Nevo Pigmentado/inducido químicamente , PPAR gamma/genética , PPAR gamma/metabolismo , Papiloma/inducido químicamente , Receptor alfa X Retinoide/genética , Neoplasias Cutáneas/inducido químicamente , Acetato de TetradecanoilforbolRESUMEN
Animal SWI2/SNF2 protein complexes containing either the brahma (BRM) or brahma-related gene 1 (BRG1) ATPase are involved in nucleosome remodelling and may control the accessibility of sequence-specific transcription factors to DNA. In vitro studies have indicated that BRM and BRG1 could regulate the expression of distinct sets of genes. However, as mice lacking BRM are viable and fertile, BRG1 might efficiently compensate for BRM loss. By contrast, as Brg1-null fibroblasts are viable but Brg1-null embryos die during the peri-implantation stage, BRG1 might exert cell-specific functions. To further investigate the in vivo role of BRG1, we selectively ablated Brg1 in keratinocytes of the forming mouse epidermis. We show that BRG1 is selectively required for epithelial-mesenchymal interactions in limb patterning, and during keratinocyte terminal differentiation, in which BRM can partially substitute for BRG1. By contrast, neither BRM nor BRG1 are essential for the proliferation and early differentiation of keratinocytes, which may require other ATP-dependent nucleosome-remodelling complexes. Finally, we demonstrate that cell-specific targeted somatic mutations can be created at various times during the development of mouse embryos cell-specifically expressing the tamoxifen-activatable Cre-ER(T2) recombinase.
Asunto(s)
Ectodermo/metabolismo , Extremidades/embriología , Queratinocitos/metabolismo , Morfogénesis , Mutagénesis/genética , Proteínas Nucleares/metabolismo , Piel/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Tipificación del Cuerpo , Diferenciación Celular , Línea Celular , ADN Helicasas , Ectodermo/citología , Células Epidérmicas , Epidermis/embriología , Epidermis/fisiología , Regulación del Desarrollo de la Expresión Génica , Queratinocitos/citología , Ratones , Ratones Noqueados , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Permeabilidad , Piel/citología , Piel/embriología , Factores de Tiempo , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
TFIID, composed of the TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs), plays a role in nucleating the assembly of the RNA polymerase II preinitiation complexes on protein coding genes. TAF10 (formerly TAF(II)30) is shared between TFIID and other transcription regulatory complexes (i.e. SAGA, TFTC, STAGA and PCAF/GCN5). TAF10 is an essential transcription factor during very early stages of mouse embryo development. To study the in vivo function of TAF10 in cellular differentiation and proliferation at later stages, the role of TAF10 was analysed in keratinocytes during skin development and adult epidermal homeostasis. We demonstrate that ablation of TAF10 in keratinocytes of the forming epidermis affects the expression of some, but not all genes, impairs keratinocyte terminal differentiation and alters skin permeability barrier functions. In contrast, loss of TAF10 in keratinocytes of adult epidermis did not (i) modify the expression of tested genes, (ii) affect epidermal homeostasis and (iii) impair acute response to UV irradiation or skin regeneration after wounding. Thus, this study demonstrates for the first time a differential in vivo requirement for a mammalian TAF for the regulation of gene expression depending on the cellular environment and developmental stage of the cell.