Hans Törmä

Changes in skin barrier function following long-term treatment with moisturizers, a randomized controlled trial

Background  Moisturizers are commonly used by patients with dry skin conditions as well as people with healthy skin. Previous studies on short‐term treatment have shown that moisturizers can weaken or strengthen skin barrier function and also influence skin barrier recovery. However, knowledge of the effects on skin barrier function of long‐term treatment with moisturizers is still scarce.

Increased expression of inducible nitric oxide synthase in psoriatic skin and cytokine-stimulated cultured keratinocytes

Summary Since nitric oxide (NO) has been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of constitutive nitric oxide synthase (cNOS) and inducibie nitric oxide synthase (iNOS) were investigated in psoriatic skin by reverse transcriptase coupled to the polymerase chain reaction (PCR). The study showed that the mRNA expression of brain nitric oxide synthase (bNOS), one of two isoforms of cNOS, was weak in both psoriatic plaques lesions and uninvolved skin, while mRNA transcripts for the second isoform, endothelial nitric oxide synthase (eNOS). were not detectable using the present method. In contrast, the mRNA expression of iNOS was markedly increased in lesional skin as compared to uninvolved skin. Cultured human keratinocytes exposed to a combination of interleukin‐lβ (IL‐1β) and tumour necrosis factor‐α (TNF‐α) for 4 h. showed strong gene expression of iNOS, while in 24h. the expression had returned to baseline expression. In summary, the study demonstrates that mRNA for the inducible form of NOS is over‐expressed in psoriatic lesions. The cause of this may be the local presence of inflammatory cytokines. These findings imply that iNOS may play an important part in local regulation of NO synthesis in psoriasis and other inflammatory dermatoses.

Protein Kinase C-Dependent Upregulation of miR-203 Induces the Differentiation of Human Keratinocytes

Terminal differentiation of keratinocytes is a multistep process that requires a coordinated program of gene expression. We aimed to explore the possible involvement of a previously unreported class of non-coding RNA genes, microRNAs (miRNAs) in keratinocyte differentiation by using miRNA expression profiling. Out of 365 miRNAs tested, 7 showed significant change between keratinocytes cultured in low or high calcium concentration. The highest-ranked upregulated gene was miR-203, whose expression was significantly upregulated in response to calcium and other inducers of keratinocyte differentiation such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and vitamin D(3). Differentiation-induced upregulation of miR-203 expression was blocked by treatment with specific inhibitors of protein kinase C (PKC), GF109203X, and Ro31-8220. Moreover, our results showed that the activator protein-1 (AP-1) proteins c-Jun and JunB regulate miR-203 expression in keratinocytes. In contrast to inducers of keratinocyte differentiation, epidermal growth factor and keratinocyte growth factor suppressed miR-203 expression in keratinocytes below the basal level. Overexpression of miR-203 in keratinocytes resulted in enhanced differentiation, whereas inhibition of miR-203 suppressed calcium-induced terminal differentiation as judged by involucrin expression. These results suggest that upregulation of miR-203 in human keratinocytes is required for their differentiation and is dependent on the activation of the PKC/AP-1 pathway.

Mutations in the Fatty Acid Transport Protein 4 Gene Cause the Ichthyosis Prematurity Syndrome

Ichthyosis prematurity syndrome (IPS) is an autosomal-recessive disorder characterized by premature birth and neonatal asphyxia, followed by a lifelong nonscaly ichthyosis with atopic manifestations. Here we show that the gene encoding the fatty acid transport protein 4 (FATP4) is mutated in individuals with IPS. Fibroblasts derived from a patient with IPS show reduced activity of very long-chain fatty acids (VLCFA)-CoA synthetase and a specific reduction in the incorporation of VLCFA into cellular lipids. The human phenotype is consistent with Fatp4 deficiency in mice that is characterized by a severe skin phenotype, a defective permeability barrier function, and perturbed VLCFA metabolism. Our results further emphasize the importance of fatty acid metabolism for normal epidermal barrier function illustrated by deficiency of a member in the FATP family of proteins.

Increased mRNA expression of manganese superoxide dismutase in psoriasis skin lesions and in cultured human keratinocytes exposed to IL-1 beta and TNF-alpha

Because reactive oxygen species have been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of the antioxidant enzyme superoxide dismutase was studied in psoriatic skin tissue. By using reverse transcription-PCR we found similar expression of copper, zinc superoxide dismutase (CuZnSOD) in the involved vs. uninvolved psoriatic skin. In contrast, the level of the manganese superoxide dismutase (MnSOD) mRNA message was consistently higher in lesional psoriatic skin as compared to adjacent uninvolved skin and healthy control skin. Parallel investigation of those cytokines that are thought to be direct or indirect inducers of the MnSOD activity revealed an increased mRNA expression of IL-1 beta, TNF-alpha, and GM-CSF in lesional psoriatic skin. To study if these cytokines exert a direct effect on dismutase expression in epidermal cells, human keratinocytes in culture were challenged with IL-1 beta, TNF-alpha, and GM-CSF. It was found that IL-1 beta and TNF-alpha, but not GM-CSF, induced the mRNA expression of MnSOD, and an additive effect was demonstrated for the two former cytokines. Further, the expression of both CuZnSOD and MnSOD transcripts was similar in cultured keratinocytes maintained at low differentiation (low Ca2+ medium) and cells forced to terminal differentiation (by high Ca2+ medium). Our results indicate that the abnormal expression of MnSOD mRNA in lesional psoriatic skin is not directly linked to the pathologic state of keratinocyte differentiation in the skin. It seems more likely that the cutaneous overexpression of MnSOD in psoriatic epidermis represents a protective cellular response evoked by cytokines released from inflammatory cells invading the diseased skin.

Chemical Chaperones Protect Epidermolysis Bullosa Simplex Keratinocytes from Heat Stress–Induced Keratin Aggregation: Involvement of Heat Shock Proteins and MAP Kinases

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by mutations in keratin genes (KRT5 or KRT14), with no existing therapies. Aggregates of misfolded mutant keratins are seen in cultured keratinocytes from severe EBS patients. In other protein-folding disorders, involvement of molecular chaperones and the ubiquitin-proteasome system may modify disease severity. In this study, the effects of heat stress on keratin aggregation in immortalized cells from two patients with EBS (KRT5) and a healthy control were examined with and without addition of various test compounds. Heat-induced (43 °C, 30 minutes) aggregates were observed in all cell lines, the amount of which correlated with the donor phenotype. In EBS cells pre-exposed to proteasome inhibitor, MG132, and p38-mitogen-activated protein kinase (MAPK) inhibitor, SB203580, the proportion of aggregate-positive cells increased, suggesting a role of proteasomes and phosphorylation in removing mutated keratin. In contrast, aggregates were reduced by pretreatment with two chemical chaperones, trimethylamine N-oxide (TMAO) and 4-phenylbutyrate (4-PBA). TMAO also modulated stress-induced p38/c-jun N-terminal kinase (JNK) activation and expression of heat shock protein (HSPA1A), the latter of which colocalized with phosphorylated keratin 5 in EBS cells. Taken together, our findings suggest therapeutic targets for EBS and other keratinopathies.

Induction of CD36 by all-trans retinoic acid: retinoic acid receptor signaling in the pathogenesis of atherosclerosis

Scavenger receptors mediating the uptake of oxidized low‐density lipoproteins (oxLDL) by macrophages play a crucial role in foam cell formation during atherosclerosis. One member of this receptor family, the thrombospondin receptor CD36, has recently been shown to mediate a major part of the oxLDL‐induced aggravation of atherosclerotic lesions. Here, we show that the expression of CD36 protein and mRNA in human monocytic THP‐1 cells is increased by all‐trans retinoic acid (atRA), a derivative of the essential Vitamin A, which leads to increased uptake of oxLDL. CD3106, a specific antagonist at the retinoic acid receptor (RAR), inhibited the atRA‐induced CD36 expression, whereas the RAR‐specific agonist CD367 induced CD36 to the same degree as atRA. This indicates an RAR‐mediated CD36 induction. AtRA and oxLDL had synergistic effects in up‐regulating CD36 when in both THP‐1 cells and primary monocytes. Applying a sensitive RAR‐GAL‐4 reporter assay, we could demonstrate RAR ligands in human atherosclerotic lesions. In addition, immunohistochemistry showed RAR‐α and ‐γ in the lesions, which indicates that atRA may contribute to foam cell formation and the progress of atherosclerosis.

Positive and negative interaction of 1,25-dihydroxyvitamin D3 and the retinoid CD437 in the induction of human melanoma cell apoptosis

The natural ligands of the nuclear receptors vitamin D receptor (VDR) and retinoic acid receptor (RAR), i.e., 1α,25‐dihydroxyvitamin D3 (VD) and all‐trans retinoic acid, have important effects on the proliferation, differentiation and apoptosis of a variety of malignant cells, including melanoma. The therapeutic potential of the 2 nuclear hormones can be enhanced by the use of synthetic analogues. In this study, the 2 human melanoma cell lines WM1341 and MeWo were compared for the combined effect of VD and synthetic retinoids. Both cell lines expressed reasonable amounts of VDR, RARγ and retinoid X receptor α and differed only in the relative expression of RARα and β. From 9 functional variants of retinoids, only the RARγ‐selective retinoid CD437 showed, in both cell lines, a significant anti‐proliferative effect. In MeWo cells, but not in WM1341 cells, VD induced growth arrest but showed no synergistic interaction with the effects of CD437. In contrast, VD induced apoptosis in WM1341, but not in MeWo, cells. CD437 was a strong inducer of apoptosis in both melanoma cell lines. Parallel treatment with CD437 and VD resulted in synergistic enhancement of apoptosis in WM1341 cells, whereas a clear decrease in induction of apoptosis in MeWo cells occurred. Our results indicate that a combined treatment of melanoma with VD and selected retinoids is promising but should be adapted to individual types of tumor. Int. J. Cancer 81:467–470, 1999.

Filaggrin Genotype Determines Functional and Molecular Alterations in Skin of Patients with Atopic Dermatitis and Ichthyosis Vulgaris

Background Several common genetic and environmental disease mechanisms are important for the pathophysiology behind atopic dermatitis (AD). Filaggrin (FLG) loss-of-function is of great significance for barrier impairment in AD and ichthyosis vulgaris (IV), which is commonly associated with AD. The molecular background is, however, complex and various clusters of genes are altered, including inflammatory and epidermal-differentiation genes. Objective The objective was to study whether the functional and molecular alterations in AD and IV skin depend directly on FLG loss-of-function, and whether FLG genotype determines the type of downstream molecular pathway affected. Methods and Findings Patients with AD/IV (n = 43) and controls (n = 15) were recruited from two Swedish outpatient clinics and a Swedish AD family material with known FLG genotype. They were clinically examined and their medical history recorded using a standardized questionnaire. Blood samples and punch biopsies were taken and trans-epidermal water loss (TEWL) and skin pH was assessed with standard techniques. In addition to FLG genotyping, the STS gene was analyzed to exclude X-linked recessive ichthyosis (XLI). Microarrays and quantitative real-time PCR were used to compare differences in gene expression depending on FLG genotype. Several different signalling pathways were altered depending on FLG genotype in patients suffering from AD or AD/IV. Disease severity, TEWL and pH follow FLG deficiency in the skin; and the number of altered genes and pathways are correlated to FLG mRNA expression. Conclusions We emphasize further the role of FLG in skin-barrier integrity and the complex compensatory activation of signalling pathways. This involves inflammation, epidermal differentiation, lipid metabolism, cell signalling and adhesion in response to FLG-dependent skin-barrier dysfunction.

Vitamin A uptake by human skin in vitro

SummaryResults are presented establishing that epidermis accumulates vitamin A from serum retinolbinding protein (RBP). Strips of human breast skin (0.2–0.3-mm thick) were incubated in a serum-free medium. From the rate of glucose oxidation, the tissue was viable for at least 48 h at 32°C in 5% CO2 air. [3H]-Retinol-RBP (10-6 M) was added to the medium for 1–24 h, after which epidermis and dermis were split and separately extracted with hexane after saponification. [3H]-Retinol was isolated by high performance liquid chromatography (HPLC). Epidermis had 6–7 times higher affinity for [3H]-retinol than dermis. The uptake could be saturated by substrate and was inhibited with unlabelled retinol-RBP but not with serum albumin. Furthermore, although the uptake was temperature-dependent, it seemed independent of cellular energy production. The epidermal accumulation of [3H]-retinol was reduced by the filtering action of dermis. On the basis of these observations, an in vitro model for the delivery of vitamin A to human skin has been proposed.