Biomedical subjects

J F Welter

Publications and source records attributed to J F Welter.

13 recordsLinked to original sources

PubMed1999

CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of keratinocyte differentiation and of involucrin gene expression. In the present study we show that a CCAAT/enhancer-binding protein (C/EBP) site in the proximal regulatory region is required for the phorbol ester response. Mutation of the C/EBP site results in the loss of basal and TPA-responsive activity. Gel mobility supershift analysis shows that C/EBPalpha binding to this site is increased by TPA treatment. Moreover, cotransfection of the human involucrin reporter plasmid with C/EBPalpha increases promoter activity to an extent comparable with TPA treatment. Mutation of the C/EBP-binding site eliminates these responses. Transfection experiments using GADD153 to create C/EBP-null conditions confirm that C/EBP factors are absolutely required for promoter activity and TPA responsiveness. C/EBPbeta and C/EBPdelta inhibit both TPA- and C/EBPalpha-dependent promoter activation, indicating functional differences among C/EBP family members. These results suggest that C/EBP transcription factor activity is necessary for basal promoter activity and TPA response of the involucrin gene.

CCAAT-Enhancer-Binding Proteins↗

PubMed1998

Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.

Involucrin is a marker of keratinocyte terminal differentiation. Our previous studies show that involucrin mRNA levels are increased by the keratinocyte differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Welter, J. F., Crish, J. F., Agarwal, C., and Eckert, R. L. (1995) J. Biol. Chem. 270, 12614-12622). We now study the signaling cascade responsible for this regulation. Protein kinase C and tyrosine kinase inhibitors inhibit both the TPA-dependent mRNA increase and the TPA-dependent increase in hINV promoter activity. The relevant response element is located within the promoter proximal regulatory region and includes an AP1 site, AP1-1. Co-transfection of the hINV promoter with dominant negative forms of Ras, MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun inhibit the TPA-dependent increase. Wild type MEKK1 enhances promoter activity and the activity can be inhibited by dominant negative MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun. In contrast, wild type Raf-1, ERK1, ERK2, MEK4, or JNK1 produced no change in activity and the dominant negative forms of these kinases failed to suppress TPA-dependent transcription. Treatment with an S6 kinase (S6K) inhibitor, or transfection with constitutively active S6K produced relatively minor changes in promoter activity, ruling out a regulatory role for S6K. These results suggest that activation of involucrin transcription involves a pathway that includes protein kinase C, Ras, MEKK1, MEK3, and p38/RK. Additional pathways that transfer MEKK1 activation via MEK1 and MEK7 also may function, but the downstream targets of these kinases need to be identified. AP1 transcription factors appear to be the ultimate target of this regulation.

Base Sequence↗

PubMed1998

Characterization of human involucrin promoter distal regulatory region transcriptional activator elements-a role for Sp1 and AP1 binding sites.

Human involucrin (hINV) is an important precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of stratifying epithelia. Previous truncation and mutagenesis experiments have shown that an activator protein 1 (Ap1) site, AP1-5, located 2100bp upstream of the transcription start site, is required for optimal promoter activity. These previous studies suggest that AP1-5 is part of a distal regulatory region spanning nucleotides -2473 to -2088. In the present report, we study the distal regulatory region (DRR), which surrounds AP1-5. Our studies show that this region contains weak and strong activator elements spanning nucleotides -2473/-2216 and -2140/-2088, respectively. The strong activator element contains AP1-5 and an adjacent specificity protein 1 (Sp1) site. The AP1-5 site is absolutely required for DRR activity, as its mutation reduces transcription to basal levels. Mutagenesis studies of the AP1-5 and Sp1 sites in the presence or absence of the weak activator element indicate that the Sp1 site and the weak activator element synergistically activate the AP1-5 site-dependent transcription. The cooperation between the Sp1 and AP1-5 sites is also observed in the context of the full-length promoter. Gel mobility shift and supershift studies show that Sp1, but not Sp2, Sp3 or Sp4 binds to the Sp1 site. When the Sp1 site is mutated or the distance between the AP1-5 and Sp1 site is increased, the binding of AP1 factors to AP1-5 is markedly reduced. Surprisingly, gel shift studies suggest that activation does not require the formation of a stable AP1/Sp1/DNA ternary complex. These studies suggest that the AP1-5 site is absolutely required for transcriptional activation, that the weak activator element and Sp1 sites serve to enhance this activation, and that the Sp1 site is required for optimal AP1 factor binding at the AP1-5 site.

Base Sequence↗

PubMed1997

S100A11, S100A10, annexin I, desmosomal proteins, small proline-rich proteins, plasminogen activator inhibitor-2, and involucrin are components of the cornified envelope of cultured human epidermal keratinocytes.

The cornified envelope (CE) is an insoluble sheath of epsilon-(gamma-glutamyl)lysine cross-linked protein, which is deposited beneath the plasma membrane during keratinocyte terminal differentiation. We have probed the structure of the CE by proteolytic cleavage of purified CE fragments isolated from CEs formed spontaneously in cell culture. CNBr digestion, followed by trypsin and then proteinase K treatment released 25%, 42%, and 18%, respectively, of the CE protein. Purification and sequencing of released peptides has identified two novel CE precursors, S100A11 (S100C, calgizzarin) and S100A10 (calpactin light chain). We also sequenced peptides derived from annexin I and plasminogen activator inhibitor 2, two putative envelope precursors, as well as portions of the well established CE precursor proteins SPR1A, SPR1B, and involucrin. Many desmosomal components were identified (desmoglein 3, desmocolin A/B, desmoplakin I, plakoglobin, and plakophilin), indicating that desmosomes become cross-linked into the CE. Fragments derived from envoplakin, the recently sequenced 210-kDa membranous CE precursor protein, which also appears to be a desmosomal component, were also identified. Analysis of the pattern of peptide release following the sequential digestion indicates that S100A11 is anchored to the envelope via Gln102 and/or Lys103 at the carboxyl terminus and at Lys3, Lys23, and/or Gln22 in the amino terminus. A similar type of analysis indicates that small proline-rich proteins 1A and 1B (SPR1A and SPR1B) become cross-linked at the amino terminus (residues 1-23) and the carboxyl terminus (residues 86-89). No loricrin, cystatin A, or elafin peptides were detected.

Amino Acid Sequence↗

PubMed1997

The epidermis: genes on - genes off.

The epidermal keratinocyte stem cell is distinguished by a relatively undifferentiated phenotype and an ability to proliferate. As part of a carefully orchestrated process, the offspring of these stem cells lose the ability to proliferate and begin a process of morphologic and biochemical transformation that results in their conversion into corneocytes. This process requires the coordinated expression of a host of cellular genes. The mechanisms responsible for regulation of these genes is an area of intense interest. In keratinocytes, as in other cell types, the expression of most genes is regulated at the transcriptional level by a class of proteins called transcription factors. Transcription factors are nuclear proteins that regulate transcription by mediating the final steps in the relay of information from the cell surface to the nucleus and the gene. These factors bind to specific DNA sequence elements located within the target gene. In this brief review we summarize evidence implicating activator protein 1 (AP1), AP2, Sp1, POU domain, CCAAT enhancer binding protein, and several other transcription factors as regulators of expression of keratinocyte genes.

Animals↗

PubMed1996

Regulation of human involucrin promoter activity by POU domain proteins.

POU domain transcription factors are expressed in the epidermis and are thought to be important regulators of keratinocyte gene expression. In the present article we demonstrate that POU transcription factors suppress transcription of the human involucrin (hINV) promoter. Cotransfection of pINV-2473, a construct containing 2473 base pairs of hINV upstream sequence linked to luciferase, with POU homeodomain transcription factors Oct1, Oct2, Brn4, SCIP, Skn1a or Skn1i, results in a strong suppression of basal promoter activity. The hINV upstream region includes a consensus POU transcription factor binding site, 5'-ATGCAAAT-3', centered around nucleotide -1277. Although this site interacts with POU factors, assays of promoter activity for a series of progressive 5' end truncations demonstrate that this site is not required for POU factor-dependent transcriptional suppression. Suppression is observed with the shortest truncation construct tested, pINV-41, suggesting that this inhibition may be mediated by effects on TATA box proteins. SCIP mutants that lack transactivation or DNA binding domains were shown to suppress transcription, suggesting that the DNA binding and transactivation domains are not required for suppression. Moreover, cotransfection of the pINV-2473 with pKSM13(+)OCT, which contains a single consensus OCT binding site, results in an increase in basal promoter activity, suggesting that endogenous POU factors suppress hINV promoter activity. In addition to inhibiting basal transcription, POU transcription factors also suppress phorbol ester-stimulated hINV promoter activity. These studies suggest that suppression of hINV promoter activity does not require the amino-terminal segment of the POU factor or direct POU factor interaction with DNA and suggest that the suppression may be via indirect interaction with other proteins in the vicinity of the TATA box. Thus, involucrin joins the ranks of a small set of genes that are regulated by POU factors in an octamer binding site-independent manner.

Base Sequence↗

PubMed1996

Transcription factor regulation of epidermal keratinocyte gene expression.

The epidermis is a tissue that undergoes a very complex and tightly controlled differentiation program. The elaboration of this program is generally flawless, resulting in the production of an effective protective barrier for the organism. Many of the genes expressed during keratinocyte differentiation are expressed in a coordinate manner; this suggests that common regulatory models may emerge. The simplest model envisions a 'common regulatory element' that is possessed by all genes that are regulated together (e.g., involucrin and transglutaminase type 1). Studies to date, however, have not identified any such elements and, if anything, the available studies suggest that appropriate expression of each gene is achieved using sometime subtly and sometime grossly different mechanisms. Recent studies indicate that a variety of transcription factors (AP1, AP2, POU domain. Sp1, STAT factors) are expressed in the epidermis and, in many cases, multiple members of several families are present (e.g., AP1 and POU domain factors). The simultaneous expression of multiple members of a single transcription factor family provides numerous opportunities for complex regulation. Some studies suggest that specific members of these families interact with specific keratinocyte genes. The best studied of these families in epidermis is the AP1 family of factors. All of the known AP1 factors are expressed in epidermis [52] and each is expressed in a specific spatial pattern that suggests the potential to regulate multiple genes. It will be important to determine the role of each of these members in regulating keratinocyte gene expression. Finally, information is beginning to emerge regarding signal transduction in keratinocytes. Some of the early events in signal transduction have been identified (e.g., PLC and PKC activation, etc.) and some of the molecular targets of these pathways (e.g., AP1 transcription factors) are beginning to be identified. Eventually we can expect to elucidation of all of the steps between the interaction of the stimulating agent with its receptor and the activation of target gene expression.

Animals↗

PubMed1996

Epidermal keratinocytes - genes and their regulation.

In recent years, the human epidermal keratinocyte has been extensively studied. These studies have shown that epidermal growth factor (EGF), transforming growth factor-beta (TGFbeta), retinoids, phorbol ester, vitamin D and other agents regulate keratinocyte proliferation, differentiation, apoptosis and gene expression. We review progress in understanding the mechanisms that regulate keratinocyte structural gene expression. In most cases little is known regarding the factors that regulate gene expression in response to a physiological agent. However, the available results suggest a role for a variety of transcription factors, including STAT factors, NFkappaB, octamer site (OCT) binding proteins and activator protein 1 (AP1) factors in regulating expression of these genes. Among these transcriptional regulators, AP1 appears to play a central role. We review the current literature regarding the regulation of involucrin, loricin, transglutaminase type 1 and cytokeratin gene expression. This survey indicates that the AP1 family of transcriptional regulators is implicated in the regulation of nearly all of these genes. We also discuss recent studies which describe the distribution of the AP1 factors, c-jun, junB, junD, Fra-1,Fra-2, c-fos and fosB, in epidermis.

Journal Article↗

PubMed1995

Differential expression of the fos and jun family members c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD during human epidermal keratinocyte differentiation.

Activator protein 1 (AP1) family proteins have been implicated in the regulation of genes expressed in the epidermis. However, no comprehensive analysis of the expression patterns of the known AP1 family proteins in the human epidermis or any other terminally differentiating tissue has been performed. In the present study we describe the localization of c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD in the normal human epidermis. Each is expressed in specific epidermal layers. c-fos is localized in the nuclei of the upper spinous and granular layer cells. FosB is present in the nuclei in all layers. Fra-1 is absent from the basal layer, but is present in all other layers. Fra-2 is detected in all layers, but staining intensity is increased in the upper spinous layer. c-jun staining is limited to the granular layer, while junB and junD are present in all layers. The differentiation-dependent pattern of expression of the AP1 family members suggest an important role for these proteins in specifying the temporal and spatial pattern of gene expression during keratinocyte differentiation.

Animals↗

PubMed1995

Fos-related antigen (Fra-1), junB, and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbol ester effects on promoter activity.

Human involucrin (hINV) is a cornified envelope precursor that is specifically expressed in the suprabasal epidermal layers. We previously demonstrated that 2500 base pairs of the hINV gene upstream regulatory region confers differentiation appropriate regulation in transgenic mice. An analysis of the hINV gene sequence upstream of the transcription start site reveals five potential AP1 binding sites (AP1-1 to 5). Using reporter gene constructs in human keratinocytes, we show that the most distal (AP1-5) and most proximal (AP1-1) AP1 sites are essential for high level transcriptional activity. Simultaneous mutation of these sites reduces transcription by 80%. Gel supershift experiments indicate the interaction of these sites with Fra-1, junB, and junD. Involucrin mRNA levels increase 10-fold and promoter activity 5-11-fold when differentiation is induced by phorbol ester. Functional studies implicate AP1-1 and AP1-5 in mediating the phorbol ester-dependent increase in promoter activity. No involucrin promoter activity or involucrin mRNA was detected in 3T3 fibroblasts. We conclude that (i) two AP1 sites in the hINV promoter are important elements required for keratinocyte-specific expression, (ii) these AP1-1 sites mediate the phorbol ester-dependent increase in promoter activity, and (iii) Fra-1, junB, and junD may be important regulators of hINV expression in epidermis.

Base Sequence↗

PubMed1993

Involucrin--structure and role in envelope assembly.

Recent findings have revealed much about the structure of involucrin. These findings make it possible to propose specific models regarding the role of involucrin and the mechanism of its crosslinking as an envelope precursor. These models provide clearly testable hypotheses that are expected to provide additional insights into the mechanism of cornified envelope assembly.

Amino Acid Sequence↗

PubMed1992

Reducing the risks in pregnant teens who are very young and those with mild mental retardation.

A 3-year evaluation of The Children and Adolescent Pregnancy Project suggests that adolescents with mental retardation may be at increased risk for early pregnancy and dropping out of school. The Pregnancy Project is a school-based intervention that primarily serves black and Hispanic pregnant teens with mild to moderate mental retardation and elementary school-age pregnant teens in the same program. Outcome data were presented in terms of four common indicators of teen pregnancy programs: low birth weight, infant mortality, school drop out, and repeat pregnancy rates. Results indicate that serving pregnant adolescents with mild to moderate mental retardation (ages 11 to 19) in the same program with very young pregnant teens (ages 11 to 15) is an effective and developmentally appropriate means of reducing risks.

Adolescent↗

PubMed1990

Cyclosporin A and tissue antigen matching in bone transplantation. Fibular allografts studied in the dog.

We studied the mechanical, metabolic, and histologic properties of short-term nonvascularized cortical bone grafts in a canine fibular graft model. Sham operated nonvascularized autotransplanted and allotransplanted bones were compared. The allografts were performed between dog leukocyte antigen (DLA) class I and II matched; DLA class I and II mismatched; and cyclosporin A (CsA) treated, DLA class I and II mismatched animals. Cyclosporin was given for 1 month, and all the animals were followed for 3 months after surgery. Mechanical properties were investigated using standard torsional tests, metabolic kinetics were assessed using isotopic prelabeling techniques, and histomorphometric analysis of cross-sectional area properties and sequential fluorochrome labels were performed. Autografts were mechanically stronger and stiffer than all the types of allograft. CsA-treated, DLA-mismatched allografts performed better than matched allografts. These in turn were stronger than non-CsA-treated, mismatched allografts, which underwent nearly complete resorption. These relationships were preserved in the metabolic and histologic analyses. In this short-term animal study, although DLA matching resulted in a slight improvement in graft outcome, mismatched grafts in dogs receiving a short course of cyclosporin A fared even better.

Animals↗