AP1 factor inactivation in the suprabasal epidermis causes increased epidermal hyperproliferation and hyperkeratosis but reduced carcinogen-dependent tumor formation.

Activator protein one (AP1) (jun/fos) factors comprise a family of transcriptional regulators (c-jun, junB, junD, c-fos, FosB, Fra-1 and Fra-2) that are key controllers of epidermal keratinocyte survival and differentiation, and are important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each member is expressed in defined cell layers during epidermal differentiation, and because AP1 factors regulate competing processes (that is, proliferation, apoptosis and differentiation). We have proposed that AP1 factors function differently in basal versus suprabasal epidermis. To test this, we inactivated suprabasal AP1 factor function in mouse epidermis by targeted expression of dominant-negative c-jun (TAM67), which inactivates function of all AP1 factors. This produces increased basal keratinocyte proliferation, delayed differentiation and extensive hyperkeratosis. These findings contrast with previous studies showing that basal layer AP1 factor inactivation does not perturb resting epidermis. It is interesting that in spite of extensive keratinocyte hyperproliferation, susceptibility to carcinogen-dependent tumor induction is markedly attenuated. These novel observations strongly suggest that AP1 factors have distinct roles in the basal versus suprabasal epidermis, confirm that AP1 factor function is required for normal terminal differentiation, and suggest that AP1 factors have a different role in normal epidermis versus cancer progression.

Suprabasal expression of the human papillomavirus type 16 oncoproteins in mouse epidermis alters expression of cell cycle regulatory proteins.

Human papillomavirus (HPV) survives by reactivating DNA replication in post-mitotic cells. In the present study, we describe a mouse model of HPV-dependent disease. In these mice, DNA synthesis is activated in suprabasal keratinocytes, leading to acanthosis, parakeratosis and enhanced desquamation. The full-length E6/E7 transcript and two alternately spliced products are produced and in most lines the predominant product is E6*. In the present study, we examine the effects of E6/E7 on cell cycle regulatory protein expression. E6/E7 expression in mouse epidermis is correlated with increased levels of the p53, p21, p27, cdk2, cdk4, cdk6, cyclin D1 and cyclin E regulatory proteins. Hyperproliferation is also observed in the buccal mucosa and the tongue epithelia of E6/E7 mice, and p53 levels are markedly increased in these epithelia. These results suggest that the major changes in cell cycle regulatory protein expression are in response to the presence of E7 and that E6 has a lesser impact.

Transgenic animal models of human papillomavirus-dependent disease (Review).

Human papillomaviruses (HPVs) are DNA tumor viruses that induce hyperproliferative lesions in cutaneous and mucosal epithelia. A wide variety of studies implicate the viral E6 and E7 oncoproteins as cell immortalizing agents, and show that these proteins work, respectively, by interfering with the function of the p53 and pRb tumor suppressor genes. Most of these studies have been performed using cell culture models. However, recently, a variety of in vivo mouse model systems have been developed for the study of HPV-dependent disease. These models use tissue-specific promoters to deliver HPV oncoprotein expression to specific body sites. Using this strategy, mouse models have been designed for the study of cancer progression in epithelia, and additional models have been designed to use E6 and E7, respectively, to probe the role of p53 and pRb on tissue differentiation and function. In the present report, we summarize the literature describing these systems, and highlight some of the important findings derived from these studies.

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.

Transcription factor Sp1 activates involucrin promoter activity in non-epithelial cell types.

The gene for human involucrin (hINV) is selectively expressed in stratifying epithelial cells lining external body surfaces. Previously, we characterized the hINV promoter 5' distal regulatory region (DRR) located between nt -2473 and -2088 upstream of the transcription start site. This region is required for optimal hINV gene expression. The DRR contains weak and strong activator elements. The strong activator comprises AP1- and Sp1-binding sites that combine to drive high-level promoter expression in human keratinocytes. Here we show that the hINV promoter is expressed in a cell-specific manner in vitro and that the DRR contains elements that are partly responsible for cell-type-specific expression of hINV. hINV promoter activity is barely detectable in 3T3 fibroblasts or HEK-293 human embryonic kidney cells. Reporter plasmids containing the full-length promoter or the isolated DRR can, however, be activated in 3T3 and HEK-293 cells by co-transfection with a plasmid encoding the transcription factor Sp1. Consistently with the lower hINV promoter activity, immunoblotting studies indicate that Sp1 protein levels are lower in 3T3 and HEK-293 cells than in human epidermal keratinocytes. Increased Sp1 protein in transfected 3T3 cells and HEK-293 cells correlates with increased promoter activity. In addition, Sp1 transfection activates the expression of the endogenous gene for hINV in HEK-293 cells. These studies suggest that Sp1 might have a role in cell-specific expression of hINV.

The distal regulatory region of the human involucrin promoter is required for expression in epidermis.

Human involucrin (hINV) is a precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of stratifying squamous epithelia. The promoter distal (DRR) and proximal regulatory regions (PRR) are required for optimal in vitro expression (Welter, J. F., Crish, J. F., Agarwal, C., and Eckert, R. L. (1995) J. Biol. Chem. 270, 12614-12622; and Banks, E. B., Crish, J. F., Welter, J. F., and Eckert, R. L. (1998) Biochem. J. 331, 61-68). We now present the complete sequence of these regions and evaluate their ability to drive in vivo transcription. Transgenes containing 5000 or 2473 base pairs of upstream regulatory region drive tissue- and differentiation-appropriate expression in stratifying surface epithelia. In contrast, transgenes containing 1953, 1333, 986, or 41 base pairs of upstream regulatory region are not expressed in surface epithelia, indicating that loss of the DRR (nucleotides -2474/-1953) results in loss of expression. Fusing the isolated DRR region directly to the hINV minimal promoter restores surface epithelial expression. Sequences downstream of the transcribed gene are not required for appropriate expression. The -1953/-41 segment influences the pattern of differentiation-dependent expression. The -986/-41 region, which includes the PRR, drives expression in internal epithelia.

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.

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.

The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation.

The epidermis is a dynamic, continually renewing structure that provides the organism with a life-sustaining interface with the environment. The major cell type of the epidermis, the epidermal keratinocyte, undergoes a complex and carefully choreographed program of differentiation. Aberrations in this process result in the genesis of a variety of debilitating and life-threatening diseases. In the present paper, we discuss the keratinocyte differentiation program and the exogenous agents that regulate differentiation. We describe the marker genes that have been utilized to study the process of gene regulation in epidermis. We describe the keratin proteins and studies that have identified keratin mutations that cause epidermal disease. We present recent information on regulation of keratinocyte gene expression and attempt to summarize current knowledge on the role of transcription factors in this process. We also discuss the process of cornified envelope assembly and the structure and function of the proteins that are thought to be precursors of this structure.

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.

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.

A dual role for involucrin in the epidermis-ultrastructural localization in epidermis and hair follicle in humans and transgenic mice.

Transgenic mice expressing human involucrin (hINV) have an abnormal hair coat appearance. To understand this phenotype, we used immunoelectron microscopy to examine hINV distribution in the hair follicle and epidermis. In human epidermis, hINV is first observed in the cytoplasm of spinous and granular layer cells. In transition cells, it is equally distributed between the cytoplasm and the nascent cornified envelope, while in the corneocytes it is largely cornified envelope-associated. In transgenic mice, the pattern of expression is similar except that much more antigen appears to be present. An important finding is the presence of hINV in hair follicle structures--low levels in the hair and high levels in the inner root sheath (IRS). The distribution of hINV is comparable in human and transgenic mouse follicles; however, much higher levels are observed in the mice. In contrast, hINV is not detected in cells of the outer root sheath (ORS). Thus, hINV is differentially expressed in two related and contiguous tissues, the epidermis and the ORS. Based on these results we conclude that hINV is a specific product of differentiated IRS cells that is localized at the cell envelope, suggesting a function as a cross-linked constituent of IRS cell envelope. These results suggest that hINV may have a dual role in epidermis, as a cross-linked structural protein in the epidermal corneocyte and the hair follicle IRS.

Tissue-specific and differentiation-appropriate expression of the human involucrin gene in transgenic mice: an abnormal epidermal phenotype.

Involucrin is a precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of the epidermis and other stratifying squamous epithelia. To study involucrin gene expression and the function of involucrin, we expressed a 6 kb DNA fragment of the human involucrin gene, containing approximately 2.5 kb of upstream sequence and 0.5 kb of downstream sequence, in transgenic mice. The transgene produces a 68 kDa protein that is detected by a human involucrin-specific antibody, and is expressed in a tissue-specific and differentiation-appropriate manner (i.e., expression is confined to the suprabasal layers of the epidermis, extocervix, trachea, esophagus and conjunctiva). Soluble involucrin levels are two to four times higher in transgenic epidermal keratinocytes compared to human foreskin keratinocytes. Newborn heterozygous animals have a normal birth weight and a normal appearing epidermis and hair growth begins at 4 to 5 days of age (i.e., the same time as hair growth in non-transgenic animals). In a subpopulation of the newborn homozygous animals birth weight is reduced, the epidermis is scaly and hair growth begins late, at around 9 to 10 days of age. In addition, the hair tends to stand erect on both heterozygous and homozygous adult animals giving the appearance of diffuse alopecia. Immunofluorescent and electron microscopy localize involucrin in the hair follicle and cornified envelope, respectively. These results suggest that overexpression of involucrin may cause abnormalities in hair follicle structure/function and cornified envelope structure. These animals provide a new model for the study of cornified envelope structure and function.

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.

Nutritional and hormonal regulation of the gene for avian malic enzyme.

This paper reviews work from our laboratory on the molecular mechanisms involved in the nutritional and hormonal regulation of avian malic enzyme. The activity of hepatic malic enzyme, one of the set of "lipogenic" enzymes, is high in well-fed chickens and low in starved chickens. In chick embryo hepatocytes in culture, insulin and triiodothyronine (T3) are positive effectors and glucagon, acting via cyclic AMP, is a negative effector. Hormone concentrations in blood are consistent with insulin and T3 playing the major positive roles, and glucagon a major negative role, in regulating hepatic malic enzyme activity during the transitions between the fed and the starved states. New results indicate that insulin-like growth factor 1 also stimulates accumulation of malic enzyme. Our strategy has been to trace the intracellular signalling pathway from its distal end, altered enzyme activity, towards its proximal end, interaction of humoral factors with their appropriate cellular receptors. Nutrition- and hormone-induced changes in malic enzyme activity are due to altered concentrations of malic enzyme protein which, in turn, are due to altered rates of synthesis of malic enzyme. Synthesis of malic enzyme is controlled by regulating the level of malic enzyme mRNA which, in turn is regulated at initiation of transcription. The next step in this analysis will be to identify cis-acting sequence elements in the malic enzyme gene which bestow upon it a selective response to nutritional state and hormones. We are using transient expression systems and avian retroviral vectors to test the function of cis-acting elements involved in the regulation of transcription.

Changes in relaxin precursor messenger ribonucleic acid levels in ovaries of rats after hysterectomy and removal of conceptuses, and during the estrous cycle.

Preprorelaxin mRNA levels in the rat ovary were determined by dot hybridization of unfractionated RNA with 32P-labeled cDNA. The sharp rise in these levels occurring near midpregnancy could be prevented by hysterectomy. After the rise occurred in intact rats, hysterectomy sharply reduced the amount of messenger. Reduction of the number of conceptuses to one also reduced ovarian preprorelaxin mRNA levels. Thus, the uterus and/or its contents were necessary for both stimulation and maintenance of preprorelaxin mRNA levels. The sharp decline in ovarian preprorelaxin mRNA levels after hysterectomy was partially prevented by the administration of 17 beta-estradiol, but not by 5 alpha-dihydrotestosterone. However, 17 beta-estradiol did not stimulate the rise in ovarian preprorelaxin mRNA levels when given to pregnant rats that were hysterectomized near the time of the natural increase in preprorelaxin mRNA levels. The amount of preprorelaxin mRNA at estrus was 3.5 times greater than the proestrous value. The estrous level was reduced to about half by the time of metestrus. The amount of ovarian preprorelaxin mRNA at estrus was 200-400 times less than that from 20-day pregnant rats. The changes in ovarian preprorelaxin mRNA levels during the estrous cycle suggest a possible role for relaxin in nonpregnant rats. Because of the correlation between changes in preprorelaxin mRNA levels and the concentration of immunoreactive relaxin in the ovaries and blood, shown by others, the amount of precursor mRNA probably regulates the concentration of relaxin in the rat ovary. The agents and mechanisms involved in this regulation remain to be determined, but estrogen appears to be important for maintenance of the elevated preprorelaxin mRNA levels in the ovary.

Changes in relaxin precursor mRNA levels in the rat ovary during pregnancy.

Levels of preprorelaxin mRNA in the rat ovary during pregnancy were determined by cell-free translation and by hybridization analyses with cloned preprorelaxin cDNA. Translation of poly(A+) RNA from rat ovaries taken at different stages of pregnancy resulted in the incorporation of [35S]cysteine into two peptides, of Mr 17,500 and 20,500, that were specifically bound by anti-relaxin IgG. Both peptides also were demonstrated by translation of ovarian poly(A+) RNA that was hybrid-selected with cloned preprorelaxin cDNA, the sequence of which corresponds to the Mr 20,500 peptide. The origin of the Mr 17,500 putative precursor is not presently known. Preprorelaxin mRNA translational activities corresponded to previously reported concentrations of relaxin in rat ovaries during pregnancy. The results of hybridization analyses, both by Northern blotting of poly(A+) RNA and dot blotting of unfractionated RNA, agreed with those of translation assays. Preprorelaxin mRNA activity/concentration was low in early pregnancy, rose markedly and reached a plateau on days 15-20 (about 1-2% of total translation activity), and then fell to low levels again by day 23, the time of parturition. These findings indicate that the concentration of relaxin in the rat ovary is directly dependent on preprorelaxin mRNA levels.

Multiple drug plus radiation enhancement during 4MV irradiation of HEp 2 cells.

Cell survival fraction for HEp 2 cells, a human epidermoid carcinoma of the larynx, was determined by exposing the cells to 4 MV linear acceleration radiation. A D0 value of 140 +/- 20 rad was measured at a dose rate of 300 rad/min. The cells were also treated concurrently with partially cytotoxic drug doses of Actinomycin D (ACD) and Adriamycin (ADRM) separately and in combination with radiation in an effort to characterize possible enhanced effects for the three component reaction species. Enhancement ratios of 1.05 +/- 0.15 for the drug-drug interaction, 1.20 +/- 0.19 (ADRM-radiation) and 1.23 +/- 0.21 (ACD-radiation) for the radiation-drug combinations and 1.28 +/- 0.17 for the drug-drug-radiation combination were measured. These data suggest that an additive response occurs with each of the drugs and radiation taken individually but little further significant enhancement is seen when all reaction components are used in combination.