RESUMEN
Interactions between normal mammary epithelial cells and extracellular matrix (ECM) are important for mammary gland homeostasis. Loss of interactions between ECM and normal mammary epithelial cells are thought to be an early event in mammary carcinogenesis. CREB-binding protein (CBP) is an important regulator of proliferation and apoptosis but the role of CBP in ECM signaling is poorly characterized. CBP was suppressed in basal-cytokeratin-positive HMECs (CK5/6+, CK14+, CK8-, CK18-, CK19-). Suppression of CBP resulted in loss of reconstituted ECM-mediated growth control and apoptosis and loss of laminin-5 alpha3-chain expression. Suppression of CBP in normal human mammary epithelial cells (HMECs) resulted in loss of CBP occupancy of the LAMA3A promoter and decreased LAMA3A promoter activity and laminin-5 alpha-3 chain expression. Exogenous expression of CBP in CBP-negative HMECs that have lost reconstituted ECM-mediated growth regulation and apoptosis resulted in (1) CBP occupancy of the LAMA3A promoter, (2) increased LAMA3A activity and laminin-5 alpha3-chain expression, and (3) enhancement of reconstituted ECM-mediated growth regulation and apoptosis. Similarly, suppression of laminin-5 alpha3-chain expression in HMECs resulted in loss of reconstituted ECM-mediated growth control and apoptosis. These observations suggest that loss of CBP in basal-cytokeratin-positive HMECs results in loss of reconstituted ECM-mediated growth control and apoptosis through loss of LAMA3A activity and laminin-5 alpha3-chain expression. Results in these studies may provide insight into early events in basal-type mammary carcinogenesis.
Asunto(s)
Apoptosis/fisiología , Proteína de Unión a CREB/fisiología , Proliferación Celular , Células Epiteliales/citología , Matriz Extracelular/metabolismo , Laminina/fisiología , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/fisiología , Apoptosis/genética , Proteína de Unión a CREB/antagonistas & inhibidores , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Polaridad Celular/genética , Células Cultivadas , Cromosomas Humanos Par 16/genética , Cromosomas Humanos Par 16/metabolismo , Regulación hacia Abajo/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Matriz Extracelular/patología , Matriz Extracelular/fisiología , Reordenamiento Génico/genética , Humanos , Laminina/antagonistas & inhibidores , Laminina/biosíntesis , Laminina/genética , Glándulas Mamarias Humanas/patología , Regiones Promotoras Genéticas , Unión Proteica/genética , Regulación hacia Arriba/genéticaRESUMEN
Retinoids and retinoic acid receptors (RARs) are important mediators of normal epithelial cell homeostasis. To assess the role of retinoids and RARs in regulating growth arrest and apoptosis in benign and malignant mammary epithelial cells, two model systems were developed: 1) RAR function was suppressed in retinoid-sensitive normal human mammary epithelial cells (HMECs) by the dominant-negative retinoic acid receptor, RARalpha403 (DNRAR), and 2) retinoid-resistant MCF-7 breast cancer cells were transduced with a functional RARbeta2. Inhibition of RAR function by the DNRAR in HMECs resulted in retinoid-resistance, increased proliferation, and dysregulated growth when cells were cultured in reconstituted extracellular matrix (rECM). Expression of RARbeta2 in MCF-7 cells resulted in sensitivity to retinoid-induced growth arrest and apoptosis. The CREB-binding protein (CBP) and the homologous protein p300 are tightly regulated, rate-limiting integrators of diverse signaling pathways and are recruited during retinoid-mediated transcriptional activation. The relationship between retinoid receptor expression, growth regulation, and transcriptional regulation of CBP/p300 is poorly understood. Inhibition of RAR function in HMECs by DNRAR suppressed expression of CBP/p300 and expression of RARbeta2 in MCF-7 cells promoted induction of CBP/p300 when cells were treated with 1.0 microM all-trans-retinoic acid (ATRA). These results suggest that ATRA and RARs regulate growth arrest of HMECs and modulate CBP/p300 protein expression. Since CBP and p300 are normally present in limiting amounts, their regulation by ATRA and RARs may be an important element in the control of transcriptional activation of genes regulating growth arrest and apoptosis.
