SPRR2A Expression Research Articles

Particulate matter 2.5 induces the skin barrier dysfunction and cutaneous inflammation via AhR- and T helper 17 cell-related genes in human skin tissue as identified via transcriptome analysis.

Particulate matter (PM2.5) is an environmental pollutant causing skin inflammatory diseases via epidermal barrier damage. However, the mechanism and related gene expression induced by PM2.5 remains unclear. Our aim was to determine the effect of PM2.5 on human skin tissue ex vivo, and elucidate the mechanism of T helper 17 cell-related inflammatory cytokine and skin barrier function. We verified the expression levels of gene in PM2.5-treated human skin tissue using Quantseq (3' mRNA-Seq), and Gene Ontology (GO) terms and protein-protein interaction (PPI) networks were performed. The PM2.5 treatment significantly enhanced the expression of Th 1, 2, 17 and 22 cell-related genes (cut-off value: │1.2 │ > fold change and p < 0.05). Most of all, Th17 cell-related genes are upregulated and those genes are associated with skin epidermal barrier function and Aryl hydrocarbon receptor (AhR), a xenobiotic receptor, pathway. In human keratinocyte cell lines, AhR-regulated genes (e.g. AhRR, CYP1A1, IL6 and IL36G), Th17 cell-related genes (e.g. IL17C) and epidermal barrier-related genes (e.g. SPRR2A and KRT71) are significantly increased after PM2.5. In the protein level, the secretion of IL-6 and IL-36G was increased in human skin tissue following PM2.5 treatment, and the expression of SPRR2A and KRT71 was significantly increased. PM2.5 exposure could ruin the skin epidermal barrier function via AhR- and Th17 cell-related inflammatory pathway.

Corneal epithelium in keratoconus underexpresses active NRF2 and a subset of oxidative stress-related genes.

Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis (KRT16, KRT14, SPRR1A, SPRR2A, SPRR3, TGM1 and TGM5) and in oxidative stress pathways (NRF2, HMOX1 and HMOX2), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium.

The glossopharyngeal nerve controls epithelial expression of Sprr2a and Krt13 around taste buds in the circumvallate papilla

Tastants reach the tip of taste bud cells through taste pores which are openings in the epithelium. We found Sprr2a is selectively expressed in the upper layer of the epithelium surrounding taste buds in the circumvallate papilla (CV) where the epithelium is organized into taste pores. Sprr2a is a member of a small proline-rich protein family, which is suggested to be involved in the restitution/migration phase of epithelial wound healing. The expression of Sprr2a was restricted to the upper layer and largely segregated with Ptch1 expression that is restricted to the basal side of the epithelium around the taste buds. Denervation resulted in the gradual loss of Sprr2a-expressing cells over 10 days similarly to that of taste bud cells which is in contrast to the rapid loss of Ptch1 expression. We also found that denervation caused an increase of Keratin (Krt)13 expression around taste buds that corresponded with the disappearance of Sprr2a and Ptch1 expression. Taste buds were surrounded by Krt13-negative cells in the CV in control mice. However, at 6 days post-denervation, taste buds were tightly surrounded by Krt13-positive cells. During taste bud development, taste bud cells emerged together with Krt13-negtive cells, and Sprr2a expression was increased along with the progress of taste bud development. These results demonstrate that regional gene expression surrounding taste buds is associated with taste bud formation and controlled by the innervating taste nerve.

Differential gene expression and methylation in the retinoid/PPARA pathway and of tumor suppressors may modify intestinal tumorigenesis induced by low folate in mice

Inadequate folate intake increases risk for colorectal cancer. We previously showed that low-folate diets induced intestinal tumors in BALB/c mice, but not in C57BL/6 mice. We suggested that DNA damage, altered methylation, and reduced apoptosis could contribute to tumorigenesis in this model. To identify genes involved in tumorigenesis, we compared gene expression profiles in preneoplastic intestine of BALB/c and C57BL/6 mice-fed low folate. We identified 74 upregulated and 90 downregulated genes in BALB/c compared to C57BL/6 mice. We validated decreased expression of Bcmo1 and increased expression of Aldh1a, which would be expected to upregulate the peroxisome proliferator-activated receptor alpha (PPARA) pathway, and confirmed the expected upregulation of several Ppara downstream genes. We verified, in BALB/c mice, reduced expression of Sprr2a, a gene that increases resistance to oxidative damage, and of two oncosuppressors (Bmp5 and Arntl). Low folate increased Ppara and Aldh1a1 expression, and decreased Bcmo1, Sprr2a, and Bmp5 expression in BALB/c, compared to BALB/c on control diets. Bcmo1, Ppara, and Bmp5 showed differential DNA methylation related to strain, diet, and/or Mthfr genotype. Disturbed regulation of the retinoid/PPARA pathway, which influences oxidative damage, and altered expression of tumor suppressors may contribute to intestinal tumorigenesis induced by low-folate intake.

SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity

BackgroundSmall proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of barrier epithelia to transiently acquire mesenchymal characteristics (EMT) and simultaneously quench reactive oxygen species during wound repair responses. p53 is also widely recognized as the node in cellular stress responses that inhibits EMT and triggers cell-cycle arrest, apoptosis, and cellular senescence. Since some p53-directed processes would seem to impede wound repair of barrier epithelia, we hypothesized that SPRR2A up regulation might counteract these effects and enable/promote wound repair under stressful environmental conditions.ResultsUsing a well characterized cholangiocarcinoma cell line we show that levels of SPRR2A expression, similar to that seen during stressful biliary wound repair responses, disrupts acetylation and subsequent p53 transcriptional activity. p53 deacetylation is accomplished via two distinct, but possibly related, mechanisms: 1) a reduction of p300 acetylation, thereby interfering with p300-p53 binding and subsequent p300 acetylation of K382 in p53; and 2) an increase in histone deacetylase 1 (HDAC1) mRNA and protein expression. The p300 CH3 domain is essential for both the autoacetylation of p300 and transference of the acetyl group to p53 and HDAC1 is a component of several non-p300 complexes that enhance p53 deacetylation, ubiquitination, and proteosomal degradation. HDAC1 can also bind the p300-CH3 domain, regulating p300 acetylation and interfering with p300 mediated p53 acetylation. The importance of this pathway is illustrated by showing complete restoration of p53 acetylation and partial restoration of p300 acetylation by treating SPRR2A expressing cells with HDAC1 siRNA.ConclusionUp-regulation of SPRR2A, similar to that seen during barrier epithelia wound repair responses reduces p53 acetylation by interfering with p300-p53 interactions and by increasing HDAC1 expression. SPRR2A, therefore, functions as a suppressor of p53-dependent transcriptional activity, which otherwise might impede cellular processes needed for epithelial wound repair responses such as EMT.

Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics

Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype.

Small proline-rich proteins (SPRR) function as SH3 domain ligands, increase resistance to injury and are associated with epithelial–mesenchymal transition (EMT) in cholangiocytes

Deficient biliary epithelial cell (BEC) expression of small proline-rich protein (SPRR) 2A in IL-6(-/-) mice is associated with defective biliary barrier function after bile duct ligation. And numerous gene array expression studies show SPRR2A to commonly be among the most highly up-regulated genes in many non-squamous, stressed and remodeling barrier epithelia. Since the function of SPRR in these circumstances is unknown, we tested the exploratory hypothesis that BEC SPRR2A expression contributes to BEC barrier function and wound repair. The effect of SPRR2A expression was studied in primary mouse BEC cultures; in a BEC cell line after forced overexpression of SPRR2A; and in human livers removed at the time of liver transplantation. Forced SPRR2A overexpression showed that it functions as a SH3 domain ligand that increases resistance to oxidative injury and promotes wound restitution by enhancing migration and acquisition of mesenchymal characteristics. Low confluency non-neoplastic mouse BEC cultures show a phenotype similar to the stable transfectants, as did spindle-shaped BEC participating in atypical ductular reactions in primary biliary cirrhosis. These observations suggest that SPRR2A-related BEC barrier modifications represent a novel, but widely utilized and evolutionarily conserved, response to stress that is worthy of further study.

Gene expression pattern and hormonal regulation of Small Proline-Rich Protein 2 family members in the female mouse reproductive system during the estrous cycle and pregnancy

Small proline-rich proteins (SPRR) are known to construct the cornified cell envelope (CE) in the stratified squamous epithelial cell. Their functions in the simple epithelium such as the uterine epithelium are not clear hitherto. In the present study, the mRNA expression patterns of sprr2 family members in the mouse uterus and vagina during the estrous cycle and pregnancy as well as their regulation by steroids were investigated. Using semi-quantitative RT-PCR, it was revealed that the transcripts of sprr2b, 2e and 2g genes were up-regulated in the proestrous and estrous uteri, and sprr2d was up-regulated only in the estrous uterus. In the vagina, transcription of sprr2a, 2b, 2d, 2e and 2k genes were up-regulated at the metestrous stage. Northern blot analysis demonstrated that the overall expression of sprr2 was highly up-regulated in the estrous uterus and the metestrous vagina. During pregnancy, the sprr2 mRNA in the uterus was sharply repressed from day 3 postcoitus on, and began to be induced around labor time. In situ hybridization showed that the sprr2 transcripts were localized in uterine luminal and glandular epithelial cells as well as vaginal stratified epithelial cells. In ovariectomized mice, the expression of sprr2a, 2d, 2e and 2f genes in the uterus were induced by estrogen, and the effect of estrogen on sprr2d and 2e expression could be partly abolished by progesterone. The data indicate that the sprr2 genes have unique regulation patterns in different reproductive tissues under different physiological conditions, and the encoded proteins might play diverse functions in the female reproductive system.

Small proline-rich proteins 2 are noncoordinately upregulated by IL-6/STAT3 signaling after bile duct ligation

Small proline-rich proteins 2 (SPRR2) are coordinately expressed with other epidermal differential complex (EDC) genes in the skin. They function as crosslinking proteins that form bridges between other proteins that comprise the cornified cell envelope, which is the major barrier against the environment. IL-6 is invariably produced at sites of biliary tract injury and IL-6-deficient (IL-6−/−) mice show impaired barrier function after bile duct ligation (BDL). Screening microarray analysis identified noncoordinate expression of SPRR2 as a candidate gene that is: (a) expressed in biliary epithelial cells (BEC); (b) IL-6 responsive; and (c) potentially related to biliary barrier function. Therefore, we studied in detail the regulation of BEC SPRR2A expression, in vitro; and tested the hypothesis that if BEC SPRR2 expression contributes to biliary barrier function, it should be increased after BDL in IL-6-wild type (IL-6+/+) mice and not in IL-6−/− mice. In vitro studies confirmed that IL-6/gp130-signaling, mediated primarily by signal transducer and activator of transcription 3 (STAT3), stimulated noncoordinate BEC SPRR2 expression. In vivo, noncoordinate upregulation of BEC SPRR2 expression after BDL was seen in the IL-6+/+ mice and was unrelated to squamous metaplasia. IL-6−/− mice showed deficient BEC SPRR2 expression after BDL associated with impaired barrier function, as evidenced by smaller diameters of obstructed ducts, decreased bile volume, and an inability to form ‘white bile' compared to IL-6+/+ mice at 12 weeks after BDL. IL-6 replacement therapy reversed the barrier defect in IL-6−/− mice after BDL, coincident with recovery of SPRR2A expression. SPRR2 in diseased mouse and human liver localized subjacent to the apical plasma membrane of BEC lining bile ducts, but was more diffusely expressed throughout the cytoplasm of cholangioles. In conclusion, BEC IL-6/gp130/STAT3 signaling noncoordinately upregulates BEC SPRR2 that appears to contribute to modification of the biliary barrier under conditions of stress.

Distinct Functional Interactions of Human Skn-1 Isoforms with Ese-1 during Keratinocyte Terminal Differentiation

Among the three major POU proteins expressed in human skin, Oct-1, Tst-1/Oct-6, and Skn-1/Oct-11, only the latter induced SPRR2A, a marker of keratinocyte terminal differentiation. In this study, we have identified three Skn-1 isoforms, which encode proteins with various N termini, generated by alternative promoter usage. These isotypes showed distinct expression patterns in various skin samples, internal squamous epithelia, and cultured human keratinocytes. Skn-1a and Skn-1d1 bound the SPRR2A octamer site with comparable affinity and functioned as transcriptional activators. Skn-1d2 did not affect SPRR2A expression. Skn-1a, the largest protein, functionally cooperated with Ese-1/Elf-3, an epithelial-specific transcription factor, previously implicated in SPRR2A induction. This cooperativity, which depended on an N-terminal pointed-like domain in Skn-1a, was not found for Skn-1d1. Actually, Skn-1d1 counteracted the cooperativity between Skn-1a and Ese-1. Apparently, the human Skn-1 locus encodes multifunctional protein isotypes, subjected to biochemical cross-talk, which are likely to play a major role in the fine-tuning of keratinocyte terminal differentiation.

Expression analysis and mutational screening of the epithelium-specific ets gene-1 (ESE-1) in patients with squamous anal cancer.

To investigate whether ESE-1 gene abnormalities are involved in alterations of epithelial cell differentiation in squamous anal cancer ESE-1 expression and structure were screened in six patients by reverse transcriptase-polymerase chain reaction (RT-PCR) and automated sequence analysis. The complete cDNA of isoform ESE-1b was always expressed and correctly spliced, with single nucleotide polymorphism being observed in two cases. Presence of ESE-1b point mutations was excluded. Expression of SPRR2A and ENDOA/CK8, two epithelium-specific ESE-1 target genes, were revealed by RT-PCR in all cases. This first report of expression of ESE-1, and of SPRR2A and ENDOA/CK8 (both related to terminal differentiation in different types of epithelia lining) in anal cancer excludes the hypothesis that these genes influenced carcinogenesis in our patients. Despite selecting of patients without clinical evidence of HPV infection, PCR consistently revealed HPV-16 DNA, highlighting the importance of HPV infection in anal cancer.

Opposite Effects of Ras or PKC Activation on the Expression of the SPRR2A Keratinocyte Terminal Differentiation Marker

Epidermal growth factor (EGF) enhances the expression of the keratinocyte terminal differentiation marker SPRR2A, when added to monolayers of basal keratinocytes, induced to stratify by increasing the extracellular calcium concentration. A similar stimulation is found during suspension-induced differentiation in methylcellulose. This effect, which is observed after several hours of EGF addition, is restricted to terminally differentiating keratinocytes and is dependent on PKC signaling. EGF also transiently activates the Ras signaling pathway, with a maximum induction after 10 min (Medema et al., 1994, Mol. Cell. Biol. 14, 7078–7085). The cellular effects of activated Ras were determined by transient transfection of Ha-ras(Leu-61) into normal human keratinocytes. Activated Ras completely inhibited PKC-mediated expression of SPRR2A. This inhibition is mediated via c-Jun as it is reversed by a dominant-negative c-Jun mutant (cJunΔ6/194) and c-Jun can substitute for activated Ras. The inhibitory effect is targeted to a 150-bp minimal promoter region, which is essential and sufficient for SPRR2A expression during keratinocyte terminal differentiation. This indicates that the Ras and PKC pathways, which both can be triggered by EGF, although at different time points, have opposite effects on SPRR2A gene expression.

Isolation and characterization of a novel epithelium-specific transcription factor, ESE-1, a member of the ets family.

We report here the isolation of a novel, highly tissue-restricted member of the ets transcription factor/oncogene family, ESE-1 (for epithelium-specific Ets), which has features distinct from those of any other ets-related factor. ESE-1 contains two putative DNA binding domains: an ETS domain, which is unique in that the 5' half shows relatively weak homology to known ets factors, and an A/T hook domain, found in HMG proteins and various other nuclear factors. In contrast to any known ets factors, ESE-1 is expressed exclusively in epithelial cells. ESE-1 expression is induced during terminal differentiation of the epidermis and in a primary human keratinocyte differentiation system. The keratinocyte terminal differentiation marker gene, SPRR2A, is a putative target for ESE-1, since SPRR2A expression during keratinocyte differentiation correlates with induction of ESE-1 expression, and ESE-1 binds with high affinity to and transactivates the ets binding site in the SPRR2A promoter. ESE-1 also binds to and transactivates the enhancer of the Endo A gene, a potential target for ESE-1 in simple epithelia. Due to the important role that other ets factors play in cellular differentiation, ESE-1 is expected to be a critical regulator of epithelial cell differentiation.