Keratinocyte ATP binding cassette transporter expression is regulated by ultraviolet light.

Many ATP binding cassette (ABC) transporters are important regulators of lipid homeostasis and have been implicated in keratinocyte lipid transport. Ultraviolet (UV) light exposure is a known epidermal stressor, which amongst other effects causes lipid alterations and defective lamellar body biogenesis. To elucidate the background of these lipid changes we studied the effect of UVB light on ABC transporter expression. The effect of UVB treatment on the levels of 47 known human ABC transporter mRNAs was analyzed in normal human epidermal keratinocytes. Immunoblots and promoter assays were carried out for ABCA1 and ABCG1. The mRNA levels of cholesterol transport regulators ABCA1 and ABCG1 were markedly downregulated by UVB, parallel to the lamellar ichthyosis related glucosylceramide transporter ABCA12 and the suspected sphingosine-1-phosphate and cholesterol sulfate transporter ABCC1. The long but not the short alternative splice variant of the ABCF2 was found to be markedly upregulated rapidly after UVB irradiation. Immunoblot confirmed ABCA1 and ABCG1 protein downregulation, and luciferase assays showed suppression of their promoters by UVB. These proteins mostly transport lipids, which account for the integrity of the epidermal barrier; therefore our findings on the UVB regulation of ABC transporters may explain the appearance of barrier dysfunction after UVB exposure.

Whole genome transcriptional profiling identifies novel differentiation regulated genes in keratinocytes.

Keratinocyte differentiation plays a pivotal role in the epidermal barrier. Single keratinocyte differentiation genes have already been studied, but many important constituents of this process may have been missed so far. Gene expression profiling by microarray was carried out in cultured normal human epidermal keratinocytes undergoing confluence-induced differentiation to find novel differentiation genes. Candidate gene lists were established and genes of potential dermatological interest were validated by quantitative reverse transcription polymerase chain reaction and immunohistochemical analysis. Some of these points lead to the identification of counter-regulation of heme oxygenase and biliverdin reductase as well as glutaredoxin and glutathione reductase indicative of potential novel redox signaling in differentiating human keratinocytes. Others indicate a strong concert down-regulation of interleukin-1 signaling at previously unidentified levels during keratinocyte differentiation. We believe that identified genes contribute to a more comprehensive understanding of the complicated epidermal differentiation process and lead to better understanding of dermatological diseases.

Novel sphingolipid derivatives promote keratinocyte differentiation.

Sphingolipids are important components of the water permeability barrier of the skin. Moreover, ceramides were also shown to influence keratinocyte differentiation and regulate cellular signalling. A confluence-induced differentiation model of normal human keratinocytes was established to allow evaluation of pro- and anti-differentiation effects of exogenous compounds. The effects of phytosphingosine (PS), sphingosine (SO), sphinganine (SA) and their hexanoyl (-C6), stearoyl (-C18) and salicyl (-SLC) derivatives, C12-alkylamine-salicylate (C12-SLC), salicylate (SLC) along with vitamin D3 (VD3) and retinol as control substances were tested in this system. Cytotoxicity assays were carried out to optimize the incubation conditions of compounds and whole genome expression changes were monitored by DNA-microarray on days 0, 1 and 4. Geometric means of gene expression levels of a subset of known keratinocyte differentiation-related genes were calculated from the microarray data to compare effects of the sphingolipid derivatives. Compound treatment-induced transcriptional changes were analysed by the ExPlain software (BIOBASE GmbH). Five of the assayed substances (SA, SO-C6, PS-C6, SO-SLC, PS-SLC) were found to be potent promoters of keratinocyte differentiation compared with VD3, and C12-SLC revealed potential anti-differentiation properties. ExPlain analysis found a different regulatory profile in the computed transcriptional networks of the sphingoid bases versus their -C6 and especially -SLC derivatives suggesting that the change in their keratinocyte differentiation modifying potential is due to a unique effect of the covalent attachment of the salicylic acid. Taken together, these results demonstrate the gene regulatory potential of sphingolipid species that could be valuable for dermatological or cosmetic applications.

Expression of tumor necrosis factor alpha and its receptors during cellular differentiation.

Tumor necrosis factor alpha (TNFalpha) is a potent proinflammatory cytokine also involved in cellular differentiation processes. TNFalpha and both of its receptors (TNFR1 and TNFR2) can be co-expressed on the same cell, allowing for local signaling. This study has examined the expression of all components necessary for autocrine cytokine regulation during human hematopoietic, epithelial, and mesenchymal models of cellular differentiation. Macrophage and dendritic differentiation of human peripheral blood monocytes decreased their TNFalpha and TNFR2 expression while increasing the TNFR1 mRNA. In colon epithelial cell lines (HT-29 and Caco-2) TNFalpha-, TNFR1-, and TNFR2-expression was decreased upon differentiation. No changes, however, were seen during human skin keratinocyte differentiation. TNFR1 expression was unchanged in all three mesenchymal lineages (adipogenesis, chondrogenesis, osteogenesis) tested. Differentiation decreases the TNFalpha message in adipocytes and the TNFR2 mRNA in adipocytes and osteocytes. Our results demonstrate that there is no general principle for TNFalpha signaling during conversion of cells from progenitor to a more differentiated phenotype. Paracrine signaling by TNFalpha to orchestrate different cell types during tissue development and remodeling, therefore, probably overrides the autocrine regulation of differentiation by TNFalpha. Non-signaling TNF-receptors may protect chondrocytes and osteocytes from the anti-differentiation effects of local TNFalpha production.

Weight loss and the renin-angiotensin-aldosterone system.

The renin-angiotensin-aldosterone system has been causally implicated in obesity-associated hypertension. We studied the influence of obesity and weight reduction on the circulating and adipose tissue renin-angiotensin-aldosterone system in menopausal women. Blood samples were analyzed for angiotensinogen, renin, aldosterone, angiotensin-converting enzyme activity, and angiotensin II. In adipose tissue biopsy samples, we analyzed angiotensinogen, renin, renin-receptor, angiotensin-converting enzyme, and angiotensin II type-1 receptor gene expression. Obese women (n=19) had higher circulating angiotensinogen, renin, aldosterone, and angiotensin-converting enzyme than lean women (n=19), and lower angiotensinogen gene expression in adipose tissue. Seventeen women successfully participated in a weight reduction protocol over 13 weeks to reduce daily caloric intake by 600 kcal. Body weight was reduced by -5%, as were angiotensinogen levels by -27%, renin by -43%, aldosterone by -31%, angiotensin-converting enzyme activity by -12%, and angiotensinogen expression by -20% in adipose tissue (all P<0.05). The plasma angiotensinogen decrease was highly correlated with the waist circumference decline (r=0.74; P<0.001). Weight and renin-angiotensin-aldosterone system reductions were accompanied by a -7-mm Hg reduced systolic ambulatory blood pressure. These data suggest that a 5% reduction in body weight can lead to a meaningfully reduced renin-angiotensin-aldosterone system in plasma and adipose tissue, which may contribute to the reduced blood pressure.

The adipose-tissue renin-angiotensin-aldosterone system: role in the metabolic syndrome?

Overfeeding of rodents leads to increased local formation of angiotensin II due to increased secretion of angiotensinogen from adipocytes. Whereas angiotensin II promotes adipocyte growth and preadipocyte recruitment, increased secretion of angiotensinogen from adipocytes also directly contributes to the close relationship between adipose-tissue mass and blood pressure in mice. In contrast, angiotensin II acts as an antiadipogenic substance in human adipose tissue, and the total increase in adipose-tissue mass may be more important in determining human plasma angiotensinogen levels than changes within the single adipocyte. However, as increased local formation of angiotensin II in adipose tissue may be increased especially in obese hypertensive subjects, a contribution of the adipose-tissue renin-angiotensin system to the development of insulin resistance and hypertension is conceivable in humans, but not yet proven. Insulin resistance may be aggravated by the inhibition of preadipocyte recruitment, which results in the redistribution of triglycerides to the liver and skeletal muscle, and blood pressure may be influenced by local formation of angiotensin II in perivascular adipose tissue. Thus, although the mechanisms are still speculative, the beneficial effects of ACE-inhibition and angiotensin-receptor blockade on the development of type 2 diabetes in large clinical trials suggest a pathophysiological role of the adipose-tissue renin-angiotensin system in the metabolic syndrome.

Human adipose tissue cells keep tight control on the angiotensin II levels in their vicinity.

Human adipose tissue expresses all components necessary for the local production of angiotensin II, which has multiple functions in adipose tissue, ranging from regulation of local blood flow to complex influences on tissue homeostasis. Still the mechanisms controlling human adipose tissue angiotensin II concentrations are not yet known. We investigated whether angiotensin II is degraded by human primary cultured preadipocytes and adipocytes and which enzymes are responsible for its metabolism. Distinct but transient angiotensin II production was limited by degradation due to consecutive proteolytic cleavage by endopeptidase and aminopeptidase activities. The endopeptidase could be identified as neprilysin expressed on the surface of both preadipocytes and adipocytes. Degradation of angiotensin II was preceded by a lag phase that was considerably longer in preadipocytes. This time span could not be explained by an induction of neprilysin nor by an increase in its surface localization. Following the lag phase, adipocytes showed a higher degradation activity than preadipocytes as mirrored by increased neprilysin levels and activity measured in their membrane fractions. Our findings demonstrate that human preadipocytes and adipocytes differentially express functional neprilysin and aminopeptidase activity involved in the regulation of angiotensin II concentrations in human adipose tissue.

Cross talk between adipose tissue cells: impact on pathophysiology.

The metabolic functionality of adipose tissue is intimately dependent on local communication between various cell types. It influences not only the equilibrium between lipogenesis and lipolysis but also between hypertrophic and hyperplastic growth, thereby determining the role adipose tissue plays in the insulin resistance syndrome.