Search PubMed⌕ Search

Biomedical subjects

R C Smart

Publications and source records attributed to R C Smart.

At least 19 recordsLinked to original sources
PubMed2006

Decreased survival of C/EBP beta-deficient keratinocytes is due to aberrant regulation of p53 levels and function.

Recent studies have identified roles for C/EBPbeta in cellular survival and tumorigenesis, however, the mechanisms through which C/EBPbeta regulates these processes are not fully understood. Previously, we demonstrated that C/EBPbeta(-/-) mice are resistant to carcinogen-induced skin tumorigenesis and in response to topical carcinogen treatment display a 17-fold increase in keratinocyte apoptosis compared to wild-type mice. Here, we have investigated the mechanisms through which C/EBPbeta regulates apoptosis in response to carcinogenic stress. Analysis of carcinogen-treated C/EBPbeta(-/-) mouse skin revealed a striking increase in the number of p53 immunopositive keratinocytes in the epidermis of C/EBPbeta(-/-) mice compared to wild-type mice and this increase was temporally associated with a concomitant anomalous increase in apoptosis. The increased levels of p53 were functional as Mdm2, Bcl-2, C/EBPalpha and p21 were differentially regulated in the epidermis of carcinogen-treated C/EBPbeta(-/-) mice. The increase in p53 protein was not associated with an increase in p53 mRNA levels. To determine whether p53 is required for the increased apoptosis in C/EBPbeta(-/-) mice, C/EBPbeta/p53 compound knockout mice were generated. Carcinogen-treated C/EBPbeta/p53 compound knockout mice did not display increased apoptosis demonstrating p53 is required for the proapoptotic phenotype in C/EBPbeta(-/-) mice. Our results demonstrate that altered keratinocyte survival in C/EBPbeta(-/-) mice results from aberrant regulation of p53 protein and function and indicate C/EBPbeta has a role in the negative regulation of p53 protein levels in response to carcinogen-induced stress.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2006

Conditional ablation of C/EBP beta demonstrates its keratinocyte-specific requirement for cell survival and mouse skin tumorigenesis.

The CCAAT/enhancer binding protein beta (C/EBP beta) is implicated in the regulation of many different molecular and physiological processes. Mice with a germline deletion of C/EBP beta (C/EBP beta(-/-)) display phenotypes in a multitude of cell types and organ systems, including skin where C/EBP beta(-/-) mice exhibit increased apoptosis in epidermal keratinocytes in response to carcinogen treatment and are completely resistant to carcinogen-induced skin tumorigenesis. To determine the contribution of systemic versus cell autonomous functions of C/EBP beta to specific phenotypes, mice with a conditional 'floxed' C/EBP beta null allele were generated. Epidermal-specific deletion of C/EBP beta was achieved by Cre recombinase expression from a keratin 5 (K5) promoter. Similar to C/EBP beta(-/-) mice, K5-Cre;C/EBP beta(fl/fl) mice were completely refractory to 7,12 dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis and these mice displayed increased DMBA-induced apoptosis in epidermal keratinocytes compared to wild-type mice. In contrast, mice lacking the related gene, C/EBP delta, were not resistant to DMBA-induced skin tumorigenesis, indicating a unique role of C/EBP beta in skin tumor development. Our findings demonstrate that C/EBP beta exerts an essential, keratinocyte-intrinsic role in cell survival in response to carcinogen treatment and the elimination of C/EBP beta in keratinocytes is sufficient to confer complete resistance of the skin to chemical carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2001

Protein kinase C-alpha coordinately regulates cytosolic phospholipase A(2) activity and the expression of cyclooxygenase-2 through different mechanisms in mouse keratinocytes.

Transgenic mice (K5-PKC alpha) in which the keratin 5 promoter directs the expression of protein kinase C-alpha (PKC alpha) to epidermal keratinocytes display a 10-fold increase in PKC alpha protein in their epidermis and alterations in phorbol ester-induced cutaneous inflammation [J Cell Science 1999;112:3497-3506]. In the current study, we have used these K5-PKC alpha mice to examine the role of PKC alpha in keratinocyte phospholipid metabolism/eicosanoid production and cutaneous inflammation. Primary keratinocytes from wild-type and transgenic mice were prelabeled in culture with [(3)H]arachidonic acid (AA) and subsequently treated with TPA. Compared with wild-type keratinocytes, K5-PKC alpha keratinocytes displayed a 2-fold increase in AA release. TPA treatment resulted in the phosphorylation of cPLA(2). PKC inhibitors GF-109203X or H7, but not mitogen-activated protein/extracellular signal-regulated protein kinase (MEK) inhibitor PD 98059, could inhibit phosphorylation and AA release. Topical 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of K5-PKC alpha mice resulted in a 5-fold increase in epidermal COX-2 induction and a 2- to 3-fold increase in prostaglandin (PG) E(2) levels above that observed in TPA-treated wild-type mice. PD 98059, GF-109203X, or H7 could block cyclooxygenase-2 (COX-2) induction by TPA. Because C/EBP beta, a basic leucine zipper transcription factor, can be activated via a PKC alpha/mitogen-activated protein kinase pathway and can influence COX-2 expression, we examined whether C/EBP beta is involved in TPA-induced epidermal COX-2 expression. TPA-induced COX-2 expression was similar in C/EBP beta nullizygous and wild-type mice. In summary, our results indicate that epidermal PKC alpha coordinately regulates cPLA(2) activity and COX-2 expression resulting in increased levels of AA and PGE(2). Furthermore, PKC alpha-induced AA release and cPLA(2) phosphorylation are independent of MEK, whereas PKC alpha-induced COX-2 expression and PGE(2) production are MEK-dependent and C/EBP beta-independent events.

Animals↗
PubMed2000

17beta-estradiol and ICI-182780 regulate the hair follicle cycle in mice through an estrogen receptor-alpha pathway.

Estradiol (E(2)) applied topically twice weekly to mouse skin at doses as low as 1 nmol inhibited hair growth by blocking the transition of the hair follicle from the resting phase (telogen) to the growth phase (anagen). In contrast, application of </=10 nmol of other steroids produced limited inhibition. Topical treatment with the estrogen receptor (ER) antagonist ICI-182780 reversed the effects of E(2), and when applied alone, ICI-182780 caused a telogen-to-anagen transition. Both E(2) and ICI-182780 were highly effective at their site of application but not at distant sites, indicating the direct rather than secondary systemic nature of their effects. Western analysis detected a 65-kDa ER-alpha immunoreactive dermal protein, and Northern analysis revealed the presence of a 6.7-kb ER-alpha mRNA. A ribonuclease protection assay confirmed the presence of ER-alpha transcripts but failed to detect ER-beta transcripts. These findings implicate a skin-specific ER-alpha pathway in the regulation of the hair follicle cycle.

Administration, Topical↗
PubMed1999

Correlation between expression of peroxisome proliferator-activated receptor beta and squamous differentiation in epidermal and tracheobronchial epithelial cells.

Previously, several members of the nuclear receptor superfamily have been implicated in the regulation of epidermal differentiation. In this study, we analyze the expression of members of the PPAR nuclear receptor subfamily in relation to the process of squamous differentiation in normal human epidermal keratinocytes (NHEK), human tracheobronchial epithelial (HBE) cells and the epidermis in vivo. Our results demonstrate that induction of differentiation in NHEK by either treatment with the phorbol ester phorbol 12-myristate-13-acetate (PMA), suspension culture or confluence greatly enhances the expression of PPARbeta mRNA. Likewise, topical treatment of mouse skin with PMA results in increased PPARbeta mRNA expression in the epidermis. In addition, the induction of squamous differentiation in HBE cells was also associated with an upregulation of PPARbeta mRNA expression. Finally, in situ hybridization analysis localized PPARbeta mRNA to the suprabasal layers of normal human skin. Our results demonstrate that the expression of PPARbeta is associated with squamous differentiation suggesting a regulatory role for this receptor in the control of specific genes during this differentiation process.

Animals↗
PubMed1999

Effects of 17-beta-estradiol and ICI 182 780 on hair growth in various strains of mice.

17-beta-Estradiol (10 nmol per 200 microl acetone) applied topically twice weekly to the clipped dorsal surface of C57BL/6 or C3H female mouse skin prevented hair growth, as previously described in the CD-1 mouse strain. Twice weekly topical application of the estrogen receptor antagonist, ICI 182 780 (10nmol per 200microl acetone), induced the telogenanagen transition and produced early pigmentation appearance in skin and hair growth in C57BL/6 and C3H female mice. Whereas twice weekly topical application of 10nmol 17-beta-estradiol blocked hair growth, the intraperitoneal administration of this dose twice weekly did not block hair growth, suggesting a direct cutaneous effect of 17-beta-estradiol. We also evaluated the effect of 17-alpha-estradiol, 17-beta-estradiol, and ICI 182 780 on hair growth in male mice. As observed in female mice, 17-beta-estradiol was a potent inhibitor of hair growth and ICI 182 780 stimulated hair growth; however, unlike the results previously observed in female mice, 17-alpha-estradiol was a potent inhibitor of hair growth in male mice. These results demonstrate that (i) the route of administration of 17-beta-estradiol is critical for its ability to block hair growth; (ii) C57BL/6 and C3H mice, two commonly employed mouse strains for hair growth studies, responded to 17-beta-estradiol and ICI 182 780 in a manner similar to that described in CD-1 mice; and (iii) the hair follicles of male and female mice respond similarly to 17-beta-estradiol and ICI 182 780, but display striking sex differences in the response to 17-alpha-estradiol on hair growth.

Administration, Topical↗
PubMed1999

A multihit, multistage model of chemical carcinogenesis.

Carcinogenesis involves the accumulation of genetic changes within a single cell. Tumor promotion functions in the initial clonal expansion of an initiated cell but is generally not considered to influence later stages. To investigate whether tumor promotion can influence later stages of carcinogenesis we developed a two-hit 7, 12-dimethylbenz[a]anthracene (D) protocol designed to enrich for keratinocytes that contain at least two D-induced genetic alterations. FVB/N mice were initiated with D and promoted with 12-O-tetradecanoylphorbol-13-acetate (T) or treated with acetone (A) vehicle for 6 weeks. At 7 weeks after the start of promotion, but before visible papilloma development, groups of mice were treated with a second dose of D or A and 1 week later T promotion was resumed. D/T/A/T mice developed 2.8 papillomas/mouse and D/A/D/T mice demonstrated an additive tumor response and developed 5.8 papillomas/mouse. Importantly, D/T/D/T mice developed 12.4 papillomas/mouse, thereby demonstrating a synergistic tumor response compared with D/A/D/T and D/T/A/T mice. D/T/D/T papillomas exhibited increases in suprabasal S phase cells and keratin 13 expression when compared with D/T/A/T papillomas. D/T/D/T mice developed squamous cell carcinomas (SCCs) 10 weeks earlier than D/T/A/T mice and demonstrated a 96% malignancy incidence and 1.71 SCC/mouse compared with D/T/A/T mice, which demonstrated a 28% malignancy incidence and 0.32 SCC/mouse. Greater than 90% of D/T/A/T and D/T/D/T papillomas and SCCs contained mutant Ha-ras, while a normal Ha-ras allele persisted in all cases, indicating that a gene other than the remaining normal allele of Ha-ras was a target gene for the second D hit. These data demonstrate that: (i) promotion between the first and second hits has a profound outcome on carcinogenesis, presumably by increasing the probability that a second hit will occur in a previously initiated cell; (ii) continued promotion after the second hit is required for full expression of malignancy; (iii) the classic initiation-promotion protocol can be extended to a multihit, multistage model.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1999

C/EBPbeta modulates the early events of keratinocyte differentiation involving growth arrest and keratin 1 and keratin 10 expression.

The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBPbeta within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBP-responsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBPbeta in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBPbeta-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBPbeta-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBPbeta-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBPbeta plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression.

Animals↗
PubMed1999

Overexpression of protein kinase C-alpha in the epidermis of transgenic mice results in striking alterations in phorbol ester-induced inflammation and COX-2, MIP-2 and TNF-alpha expression but not tumor promotion.

Protein kinase Calpha (PKCalpha) is one of six PKC isoforms expressed in keratinocytes of mouse epidermis. To gain an understanding of the role of epidermal PKCalpha, we have localized its expression to specific cells of normal mouse skin and examined the effect of keratin 5 (K5) promoter directed expression of PKCalpha in transgenic mice. In normal mouse skin, PKCalpha was extensively expressed in the outer root sheath (ORS) keratinocytes of the anagen hair follicle and weakly expressed in keratinocytes of interfollicular epidermis. K5-targeted expression of PKCalpha to epidermal basal keratinocytes and follicular ORS keratinocytes resulted in a tenfold increase in epidermal PKCalpha. K5-PKCalpha mice exhibited no abnormalities in keratinocyte growth and differentiation in the epidermis. However, a single topical treatment with the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a striking inflammatory response characterized by edema and extensive epidermal infiltration of neutrophils that formed intraepidermal microabscesses in the epidermis. Compared to TPA-treated wild-type mice, the epidermis of TPA-treated K5-PKCalpha mice displayed increased expression of cyclooxygenase-2 (COX-2), the neutrophil chemotactic factor macrophage inflammatory protein-2 (MIP-2) mRNA and the proinflammatory cytokine TNFalpha mRNA but not IL-6 or IL-1alpha mRNA. To determine if K5-PKCalpha mice display an altered response to TPA-promotion, 7, 12-dimethylbenz[a]anthracene-initiated K5-PKCalpha mice and wild-type mice were promoted with TPA. No differences in papilloma incidence or multiplicity were observed between K5-PKCalpha mice and wild-type littermates. These results demonstrate that the overexpression of PKCalpha in epidermis increases the expression of specific proinflammatory mediators and induces cutaneous inflammation but has little to no effect on epidermal differentiation, proliferation or TPA tumor promotion.

Abscess↗
PubMed1998

Mandibular distraction osteogenesis: a comparison of distraction rates in the rabbit model.

The rabbit model has been used to study the effects of different rates of distraction upon mandibular bone. Bilateral distraction was performed at two different rates anterior to the molar teeth. Both experimental groups were significantly inferior to sham operated controls in terms of bone density (DEXA) and mechanical testing. No difference in new bone formation between the two distraction rates was detected by these modalities. However, histological examination revealed non-union to be more common in the rapidly distracted group. This suggests that attempts to shorten the duration of external fixation in clinical mandibular distraction osteogenesis should be by methods other than more rapid distraction.

Absorptiometry, Photon↗
PubMed1998

Expression of CCAAT/enhancer binding proteins (C/EBP) is associated with squamous differentiation in epidermis and isolated primary keratinocytes and is altered in skin neoplasms.

The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that undergo sequential changes in gene expression during differentiation. CCAAT/enhancer binding proteins (C/EBP) are members of the bZIP family of DNA binding proteins/transcription factors. Northern analysis demonstrated that C/EBPalpha, C/EBPbeta, and C/EBPdelta mRNA are expressed in mouse epidermis and their mRNA levels were generally greater than those observed in other tissues known to express high levels of C/EBP. Western analysis of isolated epidermal cell nuclei demonstrated the presence of a 42 and 30 kDa C/EBPalpha protein and 35 kDa C/EBPbeta protein. Immunohistochemical localization of C/EBPalpha and C/EBPbeta in intact interfollicular epidermis revealed that C/EBPbeta expression is exclusive to the nuclei of a three-cell cluster of suprabasal keratinocytes that is morphologically consistent with the central column of the epidermal proliferative unit, and that C/EBPalpha is expressed in the nuclei and cytoplasm of suprabasal keratinocytes and weakly expressed in a perinuclear manner in some basal keratinocytes. In squamous cell carcinomas the expression of C/EBPalpha and C/EBPbeta was greatly diminished as both the intensity of nuclear staining and the number of cells expressing C/EBPalpha and C/EBPbeta were reduced. In isolated primary mouse keratinocytes, calcium-induced differentiation was accompanied by specific temporal changes in the expression of C/EBPalpha, C/EBPbeta, and C/EBPdelta mRNA and C/EBPalpha and C/EBPbeta protein. These results implicate a role for the C/EBP family in the regulation of genes involved in or specifically expressed during the process of squamous differentiation in epidermis.

Animals↗
PubMed1997

What are the risks of diagnostic medical radiation?

It is both ethically and economically desirable to restrict the use of diagnostic medical radiation to only those who will benefit from it. However, patients should not refuse diagnostic tests based on an exaggerated estimation of the risks because most of these tests involve low doses of radiation. It is probable that the risks derived from studies of the atomic bomb survivors, who were exposed to high doses of radiation, overestimate the risks at low doses. No evidence of thyroid cancer, leukaemia or non-Hodgkin's lymphoma has been found in patients exposed to diagnostic levels of ionising radiation. For most diagnostic tests, the risks arising from the radiation exposure are too small to be observed and the benefits will almost always outweigh the risk.

Age Factors↗
PubMed1997

Evidence that mirex promotes a unique population of epidermal cells that cannot be distinguished by their mutant Ha-ras genotype.

Mirex is a potent tumor promoter in 7,1 2-dimethylbenz[a]anthracene (DMBA)-initiated female CD-1 mouse skin. Like 12-O-tetradecanoylphorbol-13-acetate (TPA), mirex promotes papillomas that have a Ha-ras mutation; however, unlike TPA promotion, mirex promotion does not involve a general hyperplastic response. We used proliferating cell nuclear antigen (PCNA) and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemical staining to further examine the proliferative capacity of mirex. The numbers of PCNA- and BrdU-positive epidermal S-phase cells were highly concordant in all treatment groups. Unlike a single application of TPA, a single application of mirex had little or no effect on the number of S-phase epidermal cells, and chronic application of mirex to mouse skin produced only minimal increases in S-phase cells. Moreover, mirex did not significantly alter the growth of BALB/MK-2 keratinocytes in media containing either 0.05 or 1.2 mM Ca++. These results suggest that mirex may have highly specific effects on the proliferation of initiated cells and support the existence of a unique mirex mechanism and/or distinct population of mirex-promotable mutant Ha-ras epidermal cells. To begin to address this issue of a distinct population of mirex-promotable mutant Ha-ras cells, we conducted a tandem experiment in which DMBA-initiated mice were treated twice weekly with a maximal promoting dose of mirex. Then, when the number of papillomas reached a plateau, these same mice were treated twice weekly with a maximal promoting dose of TPA. Mice treated with mirex developed a maximum of 6.4 papillomas/mouse. These mice were then promoted with TPA, which produced 8.9 additional papillomas/mouse for a total of 15.3 papillomas/mouse. The maximum tumor yields from other groups of mice treated with only TPA or mirex were 9.8 and 7.3 papillomas/mouse, respectively. Therefore, under these tandem conditions, tumor yields were additive, indicating that there are at least two distinct populations of mutant Ha-ras cells: one promoted by mirex and the other by TPA.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

An estrogen receptor pathway regulates the telogen-anagen hair follicle transition and influences epidermal cell proliferation.

The hair follicle is a cyclic, self renewing epidermal structure which is thought to be controlled by signals from the dermal papilla, a specialized cluster of mesenchymal cells within the dermis. Topical treatments with 17-beta-estradiol to the clipped dorsal skin of mice arrested hair follicles in telogen and produced a profound and prolonged inhibition of hair growth while treatment with the biologically inactive stereoisomer, 17-alpha-estradiol, did not inhibit hair growth. Topical treatments with ICI 182,780, a pure estrogen receptor antagonist, caused the hair follicles to exit telogen and enter anagen, thereby initiating hair growth. Immunohistochemical staining for the estrogen receptor in skin revealed intense and specific staining of the nuclei of the cells of the dermal papilla. The expression of the estrogen receptor in the dermal papilla was hair cycle-dependent with the highest levels of expression associated with the telogen follicle. 17-beta-Estradiol-treated epidermis demonstrated a similar number of 5-bromo-2'-deoxyuridine (BrdUrd) S-phase cells as the control epidermis above telogen follicles; however, the number of BrdUrd S-phase basal cells in the control epidermis varied according to the phase of the cycle of the underlying hair follicles and ranged from 2.6% above telogen follicles to 7.0% above early anagen follicles. These findings indicate an estrogen receptor pathway within the dermal papilla regulates the telogen-anagen follicle transition and suggest that diffusible factors associated with the anagen follicle influence cell proliferation in the epidermis.

Animals↗
PubMed1996

Measurement of periprosthetic bone density in hip arthroplasty using dual-energy x-ray absorptiometry. Reproducibility of measurements.

To define the precision (reproducibility) of measurement of periprosthetic bone mineral density and bone mineral content, dual-energy x-ray absorptiometry scans were obtained on 45 randomly selected patients who had had a unilateral total hip arthroplasty within the previous 3 years. The coefficients of variation of the bone mineral density in the proximal Gruen zones were 5.0 and 5.3%, corresponding to errors of 0.07 and 0.11 g/cm2. The coefficients of variation of the bone mineral density for the distal zones averaged 2.8%, with an error of 0.08 g/cm2. The coefficients of variation of the bone mineral content were 4.8 and 2.9% for the proximal and distal zones. The contralateral femur was also scanned in 32 of the patients. For the contralateral femur bone mineral density, the coefficients of variation were 5.0% for the proximal zones and 4.8% for the distal zones. The bone mineral content was 6.0% for the contralateral regions. These results imply that differences in bone mineral density greater than 0.16 g/cm2 (2 standard errors) can be reliably measured. Dual-energy x-ray absorptiometry therefore provides a highly reproducible technique for quantitatively monitoring the changes in bone density that occur after total hip arthroplasty.

Absorptiometry, Photon↗
PubMed1996

Localization and expression of cornifin-alpha/SPRR1 in mouse epidermis, anagen hair follicles, and skin neoplasms.

Recently, cornifin-alpha/SPPR1 has been identified as a putative precursor protein of the cornified cell envelope. In this study, the expression and localization of cornifin-alpha/SPPR1 was examined in untreated and tumor promoter-treated mouse skin, hair follicles, and skin neoplasms. Western analysis with antiserum (SQ37A) to a rabbit cornifin-alpha peptide or antiserum (SQ37C) to a human SPRR1 peptide demonstrated a 31-kDa immunoreactive protein in mouse epidermis and Northern analysis revealed the presence of a 1-kb mRNA. Immunohistochemical staining of mouse skin with SQ37A or SQ37C revealed intense and specific staining of the infundibulum, isthmus, and of Henle's layer of the inner root sheath of the lower anagen hair follicle and weak staining of the telogen follicle and the suprabasal layers of the epidermis. Treatment of mouse skin with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) produced a large increase in cornifin-alpha/SPRR1 protein and mRNA. Immunohistochemical localization of cornifin-alpha/SPRR1 in TPA-treated skin indicated that cornifin-alpha/SPRR1 was increased in the suprabasal epidermis but not in the follicle. sn-1,2,-didecanoylglycerol, a model lipid second messenger, produced an increase in cornifin-alpha/SPRR1 protein similar to that of TPA, while mirex, a non-phorbol ester-type promoter had no effect. Topical doses of retinoic acid did not repress TPA-induced cornifin-alpha/SPRR1 expression. Papillomas demonstrated a 10- and 100-fold increase in cornifin-alpha/SPRR1 protein and mRNA, and expression was restricted to suprabasal cells. Squamous cell carcinomas exhibited an intermediate level of cornifin-alpha protein, and expression was restricted to keratinized areas. These data indicate: i) cornifin-alpha/SPRR1 is expressed in mouse skin; ii) cornifin-alpha/SPRR1 is localized to specific areas of the anagen hair follicle with weak staining in the telogen follicle and epidermis; iii) epidermal cornifin-alpha/SPRR1 expression is induced by phorbol ester and sn-1,2-didecanoylglycerol but not mirex, and iv) papillomas and squamous cell carcinomas demonstrate a constitutive increase in cornifin-alpha/SPRR1 in differentiated areas of the neoplasms.

Animals↗
PubMed1996

Epidermal protein kinase C-beta 2 is highly sensitive to downregulation and is exclusively expressed in Langerhans cells: downregulation is associated with attenuated contact hypersensitivity.

Treatment of mice with multiple topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol resulted in a preferential decrease in epidermal protein kinase C-beta 2 (PKC-beta 2) compared with PKC-alpha as determined by western analysis. When PKC-alpha was decreased by 40%, PKC-beta 2 could no longer be detected, suggesting that PKC-beta 2 is more sensitive to downregulation, and/or specific epidermal cell types that contain PKC-beta 2 are more sensitive to TPA/diacylglycerol. To address this issue, we isolated Langerhans cells (LCs) from epidermal cell suspensions with immunomagnetic beads and an antibody to the class II major histocompatibility complex. Northern blot analysis revealed a PKC-beta 2 signal in isolated LCs that was 40-fold greater than that observed in unfractionated epidermal cells, and no PKC-beta 2 signal was detected in epidermal cells depleted of LCs, indicating that PKC-beta 2 is expressed exclusively in LCs within the epidermis. Western blot analysis confirmed the presence of PKC-beta 2 in LCs. PKC-beta 2 was highly sensitive to downregulation, because a single application of TPA resulted in a 90% loss of PKC-beta 2 within 6 h without a decrease in the number of LCs. To determine whether the decreased level of PKC-beta 2 within LCs was associated with an alteration in contact hypersensitivity, we treated mice with only a single application of TPA, and 6 hours later mice were sensitized with 2,4-dinitrofluorobenzene on the same dorsal area. Subsequent challenge revealed a 60% decrease in contact hypersensitivity in TPA-treated mice. These data indicate that (i) within the epidermis, PKC-beta 2 is highly sensitive to downregulation and is exclusively expressed in LCs, and (ii) the downregulation of PKC-beta 2 is associated with impaired LC function with respect to contact hypersensitivity.

Animals↗