Protein Kinase D In Keratinocyte Maturation

Hdl Handle:
http://hdl.handle.net/10675.2/344626
Title:
Protein Kinase D In Keratinocyte Maturation
Authors:
Dodd, M. Ernest
Abstract:
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.
Affiliation:
Department of Physiology
Issue Date:
Aug-2004
URI:
http://hdl.handle.net/10675.2/344626
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/305097688?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorDodd, M. Ernesten
dc.date.accessioned2015-02-20T16:16:30Z-
dc.date.available2015-02-20T16:16:30Z-
dc.date.issued2004-08-
dc.identifier.urihttp://hdl.handle.net/10675.2/344626-
dc.description.abstractThe 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.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/305097688?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectPKDen
dc.subjectKeratinocyteen
dc.subjectCalciumen
dc.subjectTPAen
dc.subject1,25-dihydroxyvitamin D3en
dc.titleProtein Kinase D In Keratinocyte Maturationen
dc.typeDissertationen
dc.contributor.departmentDepartment of Physiologyen
dc.description.advisorBollag, Wendy B.en
dc.description.committeeChew, Catherine; Johnson, John; Mivechi, Nahid; Hsu, Stephenen
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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