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

There is significant variability in diet-induced obesity (DIO) among humans and rodents, which has been associated with differences in intrinsic spontaneous physical activity (SPA). The orexin neuropeptides positively modulate SPA through multiple brain sites, but the effects of DIO on orexin's activity are not well understood. In this study, we tested the hypothesis that DIO sensitivity is mediated by decreased SPA and changes in the function of the orexins. As a DIO model, we used male Sprague-Dawley rats fed a high-fat (HF; 45% kcal from fat) or a low-fat (LF; 10% kcal from fat) diet for 10 wk. We measured SPA before and after HF or LF feeding and expression of orexin receptors by real-time PCR after dietary treatments. We tested DIO effects on orexin signaling by measuring SPA after injection of orexin A in the rostral lateral hypothalamus (RLH) before and after 10 wk of HF feeding. Finally, we tested whether daily orexin A RLH injections prevent DIO caused by HF feeding. Our results show that resistance to DIO is associated with an increase in SPA, SPA after injection of orexin A in RLH, and orexin receptor expression in sites that mediate orexin's effect on SPA, including RLH. We show that daily injections of orexin peptide in RLH prevent DIO without altering food intake. We estimate that the energetic cost of SPA after orexin A RLH injection accounts for approximately 61% of the extra caloric intake associated with HF intake, suggesting additional effects of orexins. In summary, our results suggest that variability in DIO sensitivity is mediated through adaptations in the activity of the orexin peptides and their receptors.

Behavioral responses to orexin, orexin receptor gene expression, and spontaneous physical activity contribute to individual sensitivity to obesity.

Humans and rodents show large variability in their individual sensitivity to diet-induced obesity (DIO), which has been associated with differences in intrinsic spontaneous physical activity (SPA). Evidence from genetic and out-bred rat obesity models shows that higher activity of the orexin peptides results in higher intrinsic SPA and protection against DIO. Based on this, we hypothesized that naturally occurring variation in SPA and orexin signaling is sufficient to drive differences in sensitivity to DIO.

High and low activity rats: elevated intrinsic physical activity drives resistance to diet-induced obesity in non-bred rats.

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.

Kelsey Boland

Hormel Institute

University of Minnesota

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

Kelsey Boland

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University of Minnesota

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

Hormel Institute