involucrin and cholesteryl-sulfate

The mouse cholesterol sulfotransferase St2b2 contributes to epidermal differentiation by biosynthesizing cholesterol sulfate (CS) from cholesterol in the epidermis. 12-O-Tetradecanoylphorbol-13-acetate (TPA) causes epidermal hyperplasia, an abnormal increase in epidermal cell numbers resulting from aberrant cell differentiation and an increase in St2b2 protein levels. The mechanisms underlying enhanced St2b2 expression and the pathophysiologic significance of the increased expression are unclear, however. To verify whether increased St2b2 levels are necessary for TPA-induced epidermal hyperplasia, the effects of St2b2-specific small hairpin RNA (St2b2-shRNA) on hyperplasia were examined in mice. St2b2-shRNA clearly suppressed TPA-induced epidermal hyperplasia and the expression of a marker of epidermal differentiation, involucrin (INV). Interestingly, treating mouse epidermal cells with tumor necrosis factor-alpha (TNFα) increased St2b2 expression. Furthermore, treatment with TNFα-siRNA or anti-TNF receptor antibodies reduced the TPA-induced enhancement of St2b2 expression. Treatment with BAY 11-7082, a specific inhibitor of nuclear factor-kappa B (NF-κB), diminished TPA-induced St2b2 expression. These results suggested that enhancement of St2b2 expression by TPA treatment occurs mainly through the TNFα-NF-κB inflammatory signaling pathway, which in turn leads to increased CS concentrations in epidermal cells and hyperplasia.

Topics: Animals; Antibodies; Cholesterol Esters; Epidermis; Female; Hyperplasia; Inflammation; Mice; Mice, Inbred Strains; NF-kappa B; Nitriles; Protein Precursors; Receptors, Tumor Necrosis Factor; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Sulfones; Sulfotransferases; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Lipids that are synthesized de novo in the epidermis, including fatty acids, oxysterols, 1,25-dihydroxyvitamin D(3), and farnesol, can regulate the differentiation of normal human keratinocytes (NHK). Cholesterol sulfate (CS), an epidermal lipid that is produced in the upper nucleated layers of the epidermis coincident with terminal differentiation, has been shown to play a role in the regulation of the late stages of keratinocyte differentiation, including formation of the cornified envelope. In the present study, we determined i) whether CS regulates involucrin (INV), an early keratinocyte differentiation marker, and ii) the mechanism by which CS regulates differentiation. mRNA and protein levels of INV, a precursor protein of the cornified envelope, increased 2- to 3-fold in NHK incubated in the presence of CS. In contrast, cholesterol had no effect on INV protein or mRNA levels. Transcriptional regulation was assessed in NHK transfected with INV promoter-luciferase constructs. CS increased luciferase reporter activity approximately 2- to 3-fold in NHK transfected with a 3.7-kb INV promoter construct. Deletional analysis revealed a CS-responsive region of the INV promoter located between bp --2452 and --1880. A 5-base pair (bp) mutation of the AP-1 site (bp --2117 to --2111) within this responsive region abolished CS responsiveness, suggesting a role for the AP-1 complex in the regulation of INV transcription by CS. Electrophoretic mobility shift analysis demonstrated increased binding of nuclear extracts isolated from CS-treated NHK to AP-1 DNA as compared with vehicle-treated controls. Incubation of the nuclear extract with the appropriate antibodies showed that the AP-1 DNA-binding complex contained Fra-1, Fra-2, and Jun D. Western blots demonstrated that CS treatment increased the levels of Fra-1, Fra-2, and Jun D, and Northern analyses revealed that CS increased mRNA levels for these same AP-1 factors. These data indicate that CS, an endogenous lipid synthesized by keratinocytes, regulates the early stages of keratinocyte differentiation, and may do so through its ability to modulate levels of AP-1 proteins. -- Hanley, K., L. Wood, D. C. Ng, S. S. He, P. Lau, A. Moser, P. M. Elias, D. D. Bikle, M. L. Williams, and K. R. Feingold. Cholesterol sulfate stimulates involucrin transcription in keratinocytes by increasing Fra-1, Fra-2, and Jun D. J. Lipid Res. 2001. 42: 390--398.

Topics: Binding Sites; Cell Differentiation; Cholesterol; Cholesterol Esters; DNA; DNA-Binding Proteins; Fos-Related Antigen-2; Gene Expression Regulation; Humans; Keratinocytes; Liver X Receptors; Male; Mutagenesis; Orphan Nuclear Receptors; Phosphoproteins; Polymerase Chain Reaction; Protein Precursors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation

The loss of transglutaminase 1 enzyme (TGase 1) activity causes lamellar ichthyosis. Recessive X-linked ichthyosis (XI) results from accumulation of excess cholesterol 3-sulfate (CSO(4)) in the epidermis but the pathomechanism how elevated epidermal CSO(4) causes ichthyosis is largely unknown. Here we provide evidence that XI is also a consequence of TGase 1 dysfunction. TGase 1 is a key component of barrier formation in keratinocytes: it participates in the cross-linking of cell envelope (CE) structural proteins, and also forms the lipid bound envelope by esterification of long chain omega-hydroxyceramides onto CE proteins. Using involucrin and an epidermal omega-hydroxyceramide analog as substrates, kinetic analyses revealed that at membrane concentrations above 4 mol %, CSO(4) caused a marked and dose-dependent inhibitory effect on isopeptide and ester bond formation. Sequencing of tryptic peptides from TGase 1-reacted involucrin showed a large increase in deamidation of substrate glutamines. We hypothesize that supraphysiological levels of CSO(4) in keratinocyte membranes distort the structure of TGase 1 and facilitate the access of water into its active site causing hydrolysis of substrate glutamine residues. Our findings provide further evidence for the pivotal role of the TGase 1 enzyme in CE formation.

Topics: Base Sequence; Cholesterol Esters; Chromatography, High Pressure Liquid; DNA Primers; Epidermis; Esterification; Glycine; Humans; Hydrolysis; Lysine; Mutation; Protein Precursors; Recombinant Proteins; Transglutaminases

An aqueous extract of Simarouba amara was studied for its activity on the differentiation of human skin keratinocytes. Submerged and air-exposed treated keratinocyte cultures displayed a more highly differentiated histoarchitecture, with presence of ultrastructural differentiated elements, than untreated controls. Immunohistochemistry of involucrin and activation of transglutaminase activity provided further evidence for the increase in corneocyte envelope formation observed ultrastructurally. Lipid analysis of air-exposed cultures revealed an increase in the cholesterol sulphate, cholesterol and ceramide contents. After 4 weeks of treatment on the hemiface of volunteers, the capacitance and transepidermal water loss evaluation revealed the potential interest of this extract for improvement of skin hydration. Electron microscopic examination of the corneocyte envelope on tape strips confirmed its actions. Taken together these data demonstrated that an aqueous extract of S. amara increases human keratinocyte differentiation.

Topics: Cell Differentiation; Cells, Cultured; Ceramides; Cholesterol; Cholesterol Esters; Humans; Immunohistochemistry; Keratinocytes; Microscopy, Electron; Phospholipids; Plant Extracts; Plant Roots; Protein Precursors; Squalene; Sterols; Transglutaminases; Triglycerides