isolation-mouse-epidermal-keratinocytes-their-vitro-clonogenic

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

University of Minnesota

Hmga2 protein, a transcription factor involved in chromatin architecture, is expressed chiefly during development, where it has many key biological functions. When expressed in adult tissues from in various organs, Hmga2 is always related to cancer development. The role of Hmga2 in skin tumorigenesis is, however, not yet understood. We demonstrated that Hmga2 can be found in non-transformed epidermis, specifically located to the membrane of keratinocytes (KCs) in epidermis. Ex vivo culture of KCs and development of skin carcinomas in DMBA and TPA mouse models was associated with translocation of the Hmga2 protein from the membrane into the nucleus, where Hmga2 induced its own expression by binding to the Hmga2 promoter. Panobinostat, an HDAC inhibitor, downregulated Hmga2 expression by preventing Hmga2 to bind its own promoter, and thus inhibiting Hmga2 promoter activity. Hmga2 translocation to the nucleus could in part be prevented by an inhibitor for ROCK1. Our findings demonstrate that upon program of benign papilloma to malignant cSCC of skin tumorigenesis, Hmga2 translocates in a ROCK-dependent manner from the membrane to the nucleus, where it serves as an autoregulatory transcription factor, causing cell transformation.

Hmga2 translocation induced in skin tumorigenesis.

We used allogeneic bone marrow transplantation (BMT) and a mouse multistage cutaneous carcinogenesis model to probe recruitment of bone marrow-derived epithelial cells (BMDECs) in skin tumors initiated with the carcinogen, dimethylbenz[a]anthracene (DMBA), and promoted with 12-O-tetradecanolyphorbol-13-acetate (TPA). BMDECs clustered in the lesional epithelium, expressed cytokeratins, proliferated, and stratified. We detected cytokeratin induction in plastic-adherent bone marrow cells (BMCs) cultured in the presence of filter-separated keratinocytes (KCs) and bone morphogenetic protein 5 (BMP5). Lineage-depleted BMCs migrated towards High Mobility Group Box 1 (HMGB1) protein and epidermal KCs in ex vivo invasion assays. Naive female mice receiving BMTs from DMBA-treated donors developed benign and malignant lesions after TPA promotion alone. We conclude that BMDECs contribute to the development of papillomas and dysplasia, demonstrating a systemic contribution to these lesions. Furthermore, carcinogen-exposed BMCs can initiate benign and malignant lesions upon tumor promotion. Ultimately, these findings may suggest targets for treatment of non-melanoma skin cancers.

Sonali Lad

Hormel Institute

University of Minnesota

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

Sonali Lad

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University of Minnesota

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

Hormel Institute