Biomedical subjects

James F Crish

Publications and source records attributed to James F Crish.

10 recordsLinked to original sources

PubMed2006

Opposing action of curcumin and green tea polyphenol in human keratinocytes.

Persistent environmental insult can convert a normal cell into a cancer cell. However, various natural chemopreventive agents called antioxidants can retard this progression. We have recently explored the effects of several chemopreventive agents, including green tea polyphenol and curcumin, on normal human keratinocyte function. Our findings suggest that a bioactive polyphenol from green tea, (-)-epigallocatechin-3-gallate (EGCG), acts to increase involucrin gene expression, suggesting that EGCG treatment enhances normal human keratinocyte differentiation. Mechanistic studies indicate that EGCG alters mitogen-activated protein kinase cascade function to activate involucrin gene transcription via a Ras, MEKK1, MEK3, ERK1/2-p38delta cascade that targets AP1 and CAATT enhancer binding protein transcription factors. These findings suggest that EGCG may inhibit disease progression by promoting keratinocyte differentiation. Parallel studies indicate that not all antioxidants produce a similar response. Curcumin, an antioxidant derived from the turmeric, antagonizes the EGCG-dependent response by interfering in this signaling pathway. These studies suggest that different antioxidant may produce antagonistic effects in tissues.

Antioxidants↗

PubMed2006

The distal and proximal regulatory regions of the involucrin gene promoter have distinct functions and are required for in vivo involucrin expression.

Involucrin is a marker of human keratinocyte differentiation. Previous studies show that the human involucrin gene promoter has two distinct regulatory regions - the proximal regulatory region (PRR) and the distal regulatory region (DRR). To study the role of these regions in vivo, we have constructed human involucrin promoter transgenic mice and monitored the impact of specific promoter mutations on involucrin gene expression. In this study, we monitor the impact of specific mutations on expression in a range of surface epithelia. We begin by confirming previous observations made in footpad epidermis by showing that the full-length involucrin promoter drives differentiation-appropriate expression in other surface epithelia, including epidermis, cervix, and esophagus. We further show that mutation of the activator protein AP1-5 site in the DRR completely eliminates transgene expression in all of these tissues. In contrast, mutation of the DRR Sp1 site reduces overall expression, but does not alter the differentiation dependence. Additional studies identify a DRR immediate suprabasal element (ISE). Deletion of the ISE results in a loss of transgene expression in the immediate suprabasal layers. Our studies also indicate that the PRR is important for appropriate transgene expression. Mutation of a PRR C/EBP (CCAAT enhancer binding protein) transcription factor binding site results in patchy/discontinuous expression. These studies suggest that AP1, Sp1, and C/EBP transcription factors are required for appropriate differentiation-dependent involucrin expression, and that the mechanism of regulation is similar in most surface epithelia.

Animals↗

PubMed2005

h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.

Calponin is an extensively studied actin-binding protein, but its function is not well understood. Among three isoforms of calponin, h2-calponin is found in both smooth muscle and non-muscle cells. The present study demonstrates that epidermal keratinocytes and fibroblast cells express significant amounts of h2-calponin. The expression of h2-calponin is cell anchorage-dependent. The levels of h2-calponin decrease when cells are rounded up and remain low when cells are prevented from adherence to a culture dish. h2-calponin expression resumes after the floating cells are allowed to form a monolayer in plastic dish. Cell cultures on polyacrylamide gels of different stiffness demonstrated that h2-calponin expression is affected by the mechanical properties of the culture matrix. When cells are cultured on soft gel that applies less traction force to the cell and, therefore, lower mechanical tension in the cytoskeleton, the level of h2-calponin is significantly lower than that in cells cultured on hard gel or rigid plastic dish. Force-expression of h2-calponin enhanced the resistance of the actin filaments to cytochalasin B treatment. Keratinocyte differentiation is accompanied by a mechanical tension-related up-regulation of h2-calponin. Lowering the tension of actin cytoskeleton by inhibiting non-muscle myosin II ATPase decreased h2-calponin expression. In contrast to the mechanical tension regulation of endogenous h2-calponin, the expression of h2-calponin using a cytomegalovirus promotor was independent of the stiffness of culture matrix. The results suggest that h2-calponin represents a novel manifestation of mechanical tension responsive gene regulation that may modify cytoskeleton function.

Actins↗

PubMed2005

An involucrin promoter AP1 transcription factor binding site is required for expression of involucrin in the corneal epithelium in vivo.

PURPOSE: Cell division of corneal limbal stem cells gives rise to transient amplifying cells that ultimately differentiate to form the multilayered corneal epithelium. The mechanisms that regulate changes in gene expression during this process are not well understood. In the present study, the involucrin gene was used as a model to study this regulation. METHODS: Regulation of human involucrin gene expression and promoter activity was assessed using in vivo transgenic mouse models and cultured primary human corneal epithelial cells. RESULTS: Human involucrin (hINV) is a structural protein that is selectively expressed in differentiating corneal epithelial cells. The results reveal that an activator protein one (AP1) DNA-binding site is essential for appropriate basal and stimulus-dependent hINV promoter activity. Mutation of this site, AP1-5, results in a loss of hINV gene expression in the corneal epithelium in vivo and in cultured corneal epithelial cells. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1 and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased AP1 factor (Fra-1 and JunB) binding to the hINV gene AP1-5 response element. CONCLUSIONS: These findings point to an essential role for AP1 transcription factors, acting through a distal regulatory region AP1-5 element, in the regulation of involucrin gene expression during corneal epithelial cell differentiation.

Adult↗

PubMed2005

Involucrin expression in the corneal epithelium: an essential role for Sp1 transcription factors.

PURPOSE: Identifying the mechanism(s) that regulate gene expression during the transition of the limbal stem cell to a differentiated superficial cell is an important area of interest in the corneal epithelium. METHODS: However, the factors that regulate gene expression during this process are not well understood. In the present study, the human involucrin (hINV) gene was used as a model to study gene expression in the corneal epithelium. Expression was studied in normal human corneal epithelial cell cultures and hINV promoter transgenic mice. RESULTS: Studies in cultured cells revealed that an Sp transcription factor-binding site, located in the upstream regulatory region of the hINV promoter, is essential for optimal hINV gene expression. Mutation of this site reduces promoter activity. Expression of Sp1 results in an Sp1-dependent increase in activity, whereas expression of dominant-negative Sp1 inhibits promoter activity. Gel mobility shift analysis showed the interaction of Sp1 and Sp3 with the Sp DNA element. Treatment of the corneal epithelial cells with 12-O-tetradecanoylphorbol-13-acetate increased hINV gene expression and this response is associated with increased nuclear factor binding of Sp1 and Sp3 to the Sp DNA response element. Promoter mutagenesis studies in transgenic mice confirmed the importance of the Sp site, as removal of this site by promoter truncation or point mutation resulted in a complete loss of in vivo corneal epithelial cell gene expression. CONCLUSIONS: These studies provide in vivo evidence that Sp transcription factor input is absolutely necessary for activation of involucrin gene expression in the differentiating corneal epithelium.

Adult↗

PubMed2004

Antioxidants regulate normal human keratinocyte differentiation.

Cancer begins with a normal cell that, due to persistent environmental insult, is transformed, via a series of progressively more insidious steps, into a cancer cell. A major goal of chemopreventive therapy is to alter the normal cell response to the environmental agent with the goal of inhibiting disease progression. (-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive green tea antioxidant that possesses remarkable cancer chemopreventive properties. We have recently explored the hypothesis that EGCG prevents cancer by promoting keratinocyte differentiation. Based on our findings, we argue that EGCG acts to enhance the differentiation of normal keratinocytes. This is a potentially important finding, as it represents a novel mechanism of disease inhibition by EGCG--cancer preventive "differentiation therapy". However, not all antioxidant chemopreventive agents work by this mechanism. Curcumin, for example, inhibits the differentiation-promoting activity of EGCG. This report discusses the mechanism of EGCG and curcumin action in regulating expression of involucrin, a marker of keratinocyte differentiation.

Anticarcinogenic Agents↗

PubMed2004

Regulation of involucrin gene expression.

The epidermis is a dynamic renewing structure that provides life-sustaining protection from the environment. The major cell type of the epidermis, the epidermal keratinocyte, undergoes a carefully choreographed program of differentiation. Alteration of these events results in a variety of debilitating and life-threatening diseases. Understanding how this process is regulated is an important current goal in biology. In this review, we summarize the literature regarding regulation of involucrin, an important marker gene that serves as a model for understanding the mechanisms that regulate the differentiation process. Current knowledge describing the role of transcription factors and signaling cascades in regulating involucrin gene expression are presented. These studies describe a signaling cascade that includes the novel protein kinase C isoforms, Ras, MEKK1, MEK3, and a p38delta-extracellular signal regulated kinase 1/2 complex. This cascade regulates activator protein one, Sp1, and CCATT/enhancer-binding protein transcription factor DNA binding to two discrete involucrin promoter regions, the distal- and proximal-regulatory regions, to regulate involucrin gene expression.

Gene Expression Regulation↗

PubMed2003

p38 Mitogen-activated protein kinases on the body surface--a function for p38 delta.

The p38 family of mitogen-activated protein kinases includes p38 alpha (SAPK2a, CSBP), p38 beta (SAPK2b), p38 delta (SAPK4), and p38 gamma (SAPK3/ERK6). p38 alpha and p38 beta are widely expressed p38 isoforms that are involved in regulation of cell proliferation, differentiation, development, and response to stress. Relatively less is known regarding the function of the p38 delta isoform. In this review, we discuss the role of the p38 alpha, p38 beta, and p38 gamma isoforms and then present recent findings that define a role for p38 delta as a regulator of differentiation-dependent gene expression in keratinocytes.

Animals↗

PubMed2002

The human involucrin gene contains spatially distinct regulatory elements that regulate expression during early versus late epidermal differentiation.

Human involucrin (hINV) is a keratinocyte protein that is expressed in the suprabasal compartment of the epidermis and other stratifying surface epithelia. Involucrin gene expression is initiated early in the differentiation process and is maintained until terminal cell death. The distal regulatory region (DRR) is a segment of the hINV promoter (nucleotides -2473/-1953) that accurately recapitulates the normal pattern of suprabasal (spinous and granular layer) expression in transgenic mouse epithelia. To identify sequences that mediate expression at specific stages of differentiation, we divided the DRR into two segments, a 376 nucleotide upstream region (DRR(-2473/-2100)) and a 147 nucleotide downstream region (DRR(-2100/-1953)), and evaluated the ability of these sequences to drive expression in transgenic mice. The DRR(-2473/-2100) segment drives expression at a level comparable to that observed for the DRR, but expression is restricted to the upper granular layers (i.e., no spinous layer expression). In contrast, the DRR(-2100/-1953) segment does not drive expression. However, reassembling the DRR restores the complete range of expression. These results suggest that two distinct, spatially-separate elements are required to specify the complete differentiation-dependent program of involucrin gene expression. To identify specific transcription factor binding sites involved in this regulation, we mutated an activator protein-1 binding site, AP1-5, located within DRR(-2473/-2100) segment. This site binds AP1 transcription factors present in mouse epidermal extracts, and its mutation eliminates appropriate hINV expression. This result suggests that AP1 factors participate as components of a multi-component transcription factor complex that is required for regulation.

Animals↗

PubMed2002

Keratinocyte survival, differentiation, and death: many roads lead to mitogen-activated protein kinase.

The epidermis is a dynamic and continually renewing surface that provides and maintains a life-sustaining interface with the environment. The epidermal keratinocyte, the major cell type of the epidermis, undergoes a complex and carefully choreographed program of differentiation. This process requires a balance between keratinocyte proliferation, differentiation, and apoptosis. This overview will concentrate on cascades that regulate the balance between keratinocyte cell proliferation and survival, and apoptosis and cell differentiation, with a particular emphasis on the role of the mitogen-activated protein kinase cascades. A summary of the literature suggests that extracellular regulated kinases function to promote keratinocyte proliferation and survival, whereas p38 mitogen-activated protein kinase functions to promote differentiation and apoptosis.

Animals↗