• The Mechanism of Monomethylfumarate (MMF) as an Anti-psoriatic Agent

      Helwa, Inas; Department of Physiology (2014-09)
      Psoriasis is a chronic hyperproliferative inflammatory skin disorder whose primary etiology is not well understood. Keratinocytes play a pivotal role in the pathogenesis of psoriasis. The fumaric acid ester monomethylfuamarate (MMF) is the bioactive ingredient of the anti-psoriatic drug Fumaderm©, licensed in Germany since 1994. However, the exact mechanism of action of MMF is not yet well understood. Our data showed that MMF dose-dependently inhibited proliferation in primary murine and human keratinocytes and significantly increased the protein expression of the early marker of differentiation K10 and the activity of the late marker of differentiation transglutaminase enzyme. In addition, MMF inhibited mRNA expression of IL-6, TNFα and IL-1α and inhibited the protein expression of TNFα. Recently, the role of oxidative stress in psoriasis etiology has evolved and MMF has been shown to stimulate Nrf2 and mediate its nuclear translocation in other cell types. Therefore, we examined the effect of MMF on Nrf2 expression, localization and downstream effectors in keratinocytes. Nrf2 protein expression and nuclear translocation significantly increased following MMF treatment. Moreover, MMF significantly increased the mRNA expression of the Nrf2- downstream anti-oxidative enzymes, heme oxygense-1 and peroxiredoxin-6. MMF also decreased ROS generation in keratinocytes. Aquporin3 (AQP3) is a glycerol channel expressed in keratinocytes. Earlier studies from our group as well as others have shown that AQP3 plays a role in inducing early keratinocyte differentiation and that the activity of AQP3 correlates with its membranous localization. Therefore, we examined the effect of MMF on AQP3 expression and localization. MMF increased the mRNA and protein 3 expression of AQP3. In addition, MMF stimulated membranous translocation of AQP3 and increased glycerol uptake by keratinocytes. Eventually, we wanted to examine whether Nrf2 plays a role in the expression of AQP3. Our data showed that the Nrf2 stimulator sulforaphane (SFN) increased the expression of AQP3. Thus, our data suggest that MMF exerts its action through Nrf2 stimulation. Nrf2 stimulation helps to regain keratinocyte oxidative balance and may also play a role in inducing AQP3 expression and activity. This provides the molecular basis for the MMF-mediated improvement of keratinocyte differentiation and inhibition of keratinocyte proliferation.
    • Protein Kinase D In Keratinocyte Maturation

      Dodd, M. Ernest; Department of Physiology (2004-08)
      The epidermis is important for the body's maintenance of water homeostasis and resistance to environmental stress, and the m ajor cell type of the epidermis is the keratinocyte. Keratinocyte maturation requires proliferation, followed by terminal differentiation, and diseases of the skin often exhibit deregulated epidermal maturation. Protein kinase D (PKD) expression correlates with proliferation in keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. W e have hypothesized that PKD functions as a pro-proliferative and/or anti-differentiative signal in primary mouse keratinocytes and have predicted that agents that stimulate differentiation might also initiate a reduction in PKD expression and/or activation to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation and activity were analyzed upon treatment with differentiating agents and with 1 2 -0 - tetradecanoylphorbol-13-acetate, TPA, which stimulates differentiation acutely and proliferation chronically. 1,25-dihydroxyvitamin D3 -, elevated extracellular calcium-, and acute TPA-induced differentiation down-modulated PKD levels and autophosphorylation at serine 916. In addition, elevated extracellular calcium- and acute TPA-induced differentiation down-modulated PKD activity. Chronic TPA treatment stimulated proliferation and caused a recovery o f PKD levels, autophosphorylation and activity. In co-transfection experiments in keratinocytes, co-expression of PKD increased and decreased the promoter activities of keratin 5, a marker of proliferation, and involucrin, a marker of differentiation, respectively, and opposed the effects of elevated extracellular calcium on the expression of these markers. W hile cloning PKD for expression studies, we identified a splice variant of PKD, PKD{3, which is differentially spliced in a region important in activation and subcellular localization. Therefore, we hypothesized that this splice variant may have dissimilar activation properties and/or alternate roles in keratinocyte maturation. However, in vitro activation studies demonstrated equal activation of PK D a (full length) and PKDj3 by TPA and DAG. Co-transfection experiments showed that P K D a and PKDp affected marker expression to the same degree and similarly opposed the effects of elevated extracellular calcium-induced differentiation on marker expression. Our work represents the first demonstration of: 1) down-modulation o f PKD during differentiation, 2) pro-proliferative/anti-differentiative effects of PKD on keratinocyte marker expression and 3) existence of a splice variant of PKD.
    • The Role of Phospholipase D2 and Its Interaction with Aquaporin 3 in Primary

      Qin, Haixia; Department of Physiology (2010-04)
      Regulated keratinocyte proliferation and differentiation is necessary for normal skin function. In mouse keratinocytes phospholipase D2 (PLD2) colocalizes with aquaporin 3 (AQP3), probably via a direct protein-protein interaction as seen in Sf9 cells. Since AQP3 can transport glycerol, a substrate of PLD2, we hypothesized that AQP3 and PLD2 function together to form phosphatidylglycerol (PG), a lipid second messenger which inhibits keratinocyte proliferation and promote differentiation. First, adenovirusmediated PLD2 overexpression enhanced keratinocyte proliferation under control conditions and inhibited differentiation induced by a moderately elevated calcium level. However, PG synthesis was inhibited with PLD2 overexpression; this decrease may result from disruption of the endogenous PLD2 and AQP3 interaction, and/or reduced AQP3 activity following overexpression of PLD2. Next, AQP3 was overexpressed. Overexpression of either PLD2 or AQP3 inhibited the activity of transglutaminase (TGase), a marker of keratinocyte differentiation. However, co-overexpression of AQP3 and PLD2 returned TGase activity to control levels, under both control and calciumstimulated conditions. Similarly, PG synthesis was inhibited by either PLD2 or AQP3 overexpression, but PG levels were returned to control values with co-overexpression. These results are consistent with our hypothesis that PG is a differentiation signal: less PG leads to proliferation and inhibition of differentiation. The caveolin-1 scaffolding domain peptide has been found to interact functionally with PLD2 in low-density membrane microdomains. We propose that reduced AQP3 and PLD2 interaction resulting from disruption of lipid rafts by the caveolin-1 scaffolding domain peptide results in less PG synthesis and the inhibition of calcium-induced keratinocyte differentiation. Mouse keratinocytes were treated with cell-permeable caveolin-1 scaffolding domain peptide (CSDP) and cell differentiation was stimulated using a moderately elevated extracellular calcium concentration. The CSDP had no effect itself on PG synthesis, differentiation or proliferation, but it prevented the changes induced by a moderate calcium concentration, whereas a negative control peptide did not. The CSDP altered PLD2 distribution within membrane microdomains, but had little or no effect on AQP3 distribution. Finally, we showed that the CSDP disrupted lipid rafts in cell membranes by itself, while when applied with calcium simultaneously it prevented the changes induced by moderate calcium. We conclude that the CSDP regulates both calcium-inhibited proliferation and -stimulated differentiation, at least in part, through effects on PG production.