Astrid L. Basse

RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes

RhoA is a small GTPase shown to be crucial for cytokinesis, stress fiber formation, and epithelial cell–cell contacts. Analyzing mice with a keratinocyte-restricted deletion of the RhoA gene, we find that RhoA is not required for skin development and maintenance but has specific functions in vitro.

Rac1 is crucial for Ras-dependent skin tumor formation by controlling Pak1-Mek-Erk hyperactivation and hyperproliferation in vivo.

Rac1 has a role in proliferation and survival of tumor cells in vitro. The exact effects of Rac1 on growth, apoptosis and corresponding signaling pathways during tumorigenesis in vivo, however, have not been explored yet. Using mice with a keratinocyte-restricted deletion of the Rac1 gene, we found that Rac1 is essential for DMBA/TPA-induced skin tumor formation. This corresponded to a decreased keratinocyte hyperproliferation, although apoptosis was not detectably altered. Activated Rac1 promoted Erk-dependent hyperproliferation by Pak1-mediated Mek activation independent of Mek1 phosporylation at serine 298. Rac1 was furthermore required for Pak2-dependent hyperactivation of Akt, which under in vivo condition was restricted to the suprabasal cell layers corresponding to a suprabasal-specific expression of Pak2. It is surprising that none of these signaling pathways was altered in untreated Rac1-deficient skin, indicating a hyperproliferation-specific function of Rac1 in vivo. These data suggest that blocking of Rac1 function might allow tumor-specific growth repression, as Rac1 is not required for normal growth and growth signaling controlling pathways in skin in vivo.

Transcriptome profiling of brown adipose tissue during cold exposure reveals extensive regulation of glucose metabolism

We applied digital gene expression profiling to determine the transcriptome of brown and white adipose tissues (BAT and WAT, respectively) during cold exposure. Male C57BL/6J mice were exposed to cold for 2 or 4 days. A notable induction of genes related to glucose uptake, glycolysis, glycogen metabolism, and the pentose phosphate pathway was observed in BAT from cold-exposed animals. In addition, glycerol-3-phosphate dehydrogenase 1 expression was induced in BAT from cold-challenged mice, suggesting increased synthesis of glycerol from glucose. Similarly, expression of lactate dehydrogenases was induced by cold in BAT. Pyruvate dehydrogenase kinase 2 (Pdk2) and Pdk4 were expressed at significantly higher levels in BAT than in WAT, and Pdk2 was induced in BAT by cold. Of notice, only a subset of the changes detected in BAT was observed in WAT. Based on changes in gene expression during cold exposure, we propose a model for the intermediary glucose metabolism in activated BAT: 1) fluxes through glycolysis and the pentose phosphate pathway are induced, the latter providing reducing equivalents for de novo fatty acid synthesis; 2) glycerol synthesis from glucose is increased, facilitating triacylglycerol synthesis/fatty acid re-esterification; 3) glycogen turnover and lactate production are increased; and 4) entry of glucose carbon into the tricarboxylic acid cycle is restricted by PDK2 and PDK4. In summary, our results demonstrate extensive and diverse gene expression changes related to glucose handling in activated BAT.

Retinoic acid has different effects on UCP1 expression in mouse and human adipocytes

BackgroundIncreased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes.ResultsThe effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α.ConclusionsUCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA.

N-WASP is a novel regulator of hair-follicle cycling that controls antiproliferative TGFβ pathways

N-WASP is a cytoplasmic molecule mediating Arp2/3 nucleated actin polymerization. Mice with a keratinocyte-specific deletion of the gene encoding N-WASP showed normal interfollicular epidermis, but delayed hair-follicle morphogenesis and abnormal hair-follicle cycling, associated with cyclic alopecia and prolonged catagen and telogen phases. The delayed anagen onset correlated with an increased expression of the cell-cycle inhibitor p21CIP, and increased activity of the TGFβ pathway, a known inducer of p21CIP expression. Primary N-WASP-null keratinocytes showed reduced growth compared with control cells and enhanced expression of the gene encoding the cell-cycle inhibitor p15INK4B, a TGFβ target gene. Inhibition of TGFβ signaling blocked overexpression of p15INK4B and restored proliferation of N-WASP-deficient keratinocytes in vitro. However, induction of N-WASP gene deletion in vitro did not result in obvious changes in TGFβ signaling or growth of keratinocytes, indicating that the in vivo environment is required for the phenotype development. These data identify the actin nucleation regulator N-WASP as a novel element of hair-cycle control that modulates the antiproliferative and pro-apoptotic TGFβ pathway in keratinocytes in vivo and in vitro.

ERRγ enhances UCP1 expression and fatty acid oxidation in brown adipocytes

Estrogen‐related receptors (ERRs) are important regulators of energy metabolism. Here we investigated the hypothesis that ERRγ impacts on differentiation and function of brown adipocytes.

Bioenergetic modulation with dichloroacetate reduces the growth of melanoma cells and potentiates their response to BRAFV600E inhibition

BackgroundAdvances in melanoma treatment through targeted inhibition of oncogenic BRAF are limited owing to the development of acquired resistance. The involvement of BRAFV600E in metabolic reprogramming of melanoma cells provides a rationale for co-targeting metabolism as a therapeutic approach.MethodsWe examined the effects of dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, on the growth and metabolic activity of human melanoma cell lines. The combined effect of DCA and the BRAF inhibitor vemurafenib was investigated in BRAFV600E -mutated melanoma cell lines. Vemurafenib-resistant cell lines were established in vitro and their sensitivity to DCA was tested.ResultsDCA induced a reduction in glycolytic activity and intracellular ATP levels, and inhibited cellular growth. Co-treatment of BRAFV600E-mutant melanoma cells with DCA and vemurafenib induced a greater reduction in intracellular ATP levels and cellular growth than either compound alone. In addition, melanoma cells with in vitro acquired resistance to vemurafenib retained their sensitivity to DCA.ConclusionsThese results suggest that DCA potentiates the effect of vemurafenib through a cooperative attenuation of energy production. Furthermore, the demonstration of retained sensitivity to DCA in melanoma cells with acquired resistance to vemurafenib could have implications for melanoma treatment.

Global gene expression profiling of brown to white adipose tissue transformation in sheep reveals novel transcriptional components linked to adipose remodeling.

BackgroundLarge mammals are capable of thermoregulation shortly after birth due to the presence of brown adipose tissue (BAT). The majority of BAT disappears after birth and is replaced by white adipose tissue (WAT).ResultsWe analyzed the postnatal transformation of adipose in sheep with a time course study of the perirenal adipose depot. We observed changes in tissue morphology, gene expression and metabolism within the first two weeks of postnatal life consistent with the expected transition from BAT to WAT. The transformation was characterized by massively decreased mitochondrial abundance and down-regulation of gene expression related to mitochondrial function and oxidative phosphorylation. Global gene expression profiling demonstrated that the time points grouped into three phases: a brown adipose phase, a transition phase and a white adipose phase. Between the brown adipose and the transition phase 170 genes were differentially expressed, and 717 genes were differentially expressed between the transition and the white adipose phase. Thirty-eight genes were shared among the two sets of differentially expressed genes. We identified a number of regulated transcription factors, including NR1H3, MYC, KLF4, ESR1, RELA and BCL6, which were linked to the overall changes in gene expression during the adipose tissue remodeling. Finally, the perirenal adipose tissue expressed both brown and brite/beige adipocyte marker genes at birth, the expression of which changed substantially over time.ConclusionsUsing global gene expression profiling of the postnatal BAT to WAT transformation in sheep, we provide novel insight into adipose tissue plasticity in a large mammal, including identification of novel transcriptional components linked to adipose tissue remodeling. Moreover, our data set provides a useful resource for further studies in adipose tissue plasticity.

The protein source determines the potential of high protein diets to attenuate obesity development in C57BL/6J mice

abstract The notion that the obesogenic potential of high fat diets in rodents is attenuated when the protein:carbohydrate ratio is increased is largely based on studies using casein or whey as the protein source. We fed C57BL/6J mice high fat-high protein diets using casein, soy, cod, beef, chicken or pork as protein sources. Casein stood out as the most efficient in preventing weight gain and accretion of adipose mass. By contrast, mice fed diets based on pork or chicken, and to a lesser extent mice fed cod or beef protein, had increased adipose tissue mass gain relative to casein fed mice. Decreasing the protein:carbohydrate ratio in diets with casein or pork as protein sources led to accentuated fat mass accumulation. Pork fed mice were more obese than casein fed mice, and relative to casein, the pork-based feed induced substantial accumulation of fat in classic interscapular brown adipose tissue accompanied by decreased UCP1 expression. Furthermore, intake of a low fat diet with casein, but not pork, as a protein source reversed diet-induced obesity. Compared to pork, casein seems unique in maintaining the classical brown morphology in interscapular brown adipose tissue with high UCP1 expression. This was accompanied by increased expression of genes involved in a futile cycling of fatty acids. Our results demonstrate that intake of high protein diets based on other protein sources may not have similar effects, and hence, the obesity protective effect of high protein diets is clearly modulated by protein source.

An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

ABSTRACT Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo “browning.” In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.