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1.  Small Proline Rich Proteins (SPRR) Function as SH3 Domain Ligands, Increase Resistance to Injury and are Associated with Epithelial-Mesenchymal Transition (EMT) in cholangiocytes 
Journal of hepatology  2007;48(2):276-288.
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 upregulated 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 BEC cell line after forced over expression of SPRR2A; and in human livers removed at the time of liver transplantation.
Forced SPRR2A over-expression 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.
PMCID: PMC2263141  PMID: 18155796
Cholangiocytes; Ductular Reaction; Wound Healing; Oxidative Stress; Repair; SH3 domain; EMT
2.  Involvement of a nuclear matrix association region in the regulation of the SPRR2A keratinocyte terminal differentiation marker. 
Nucleic Acids Research  1998;26(23):5288-5294.
The small proline-rich protein genes ( SPRRs ) code for precursors of the cornified cell envelope, and are specifically expressed during keratinocyte terminal differentiation. The single intron of SPRR2A enhanced the activity of the SPRR2A promoter in transient transfection assays. This enhancement was position dependent, and did not function in combination with a heterologous promoter, indicating that the intron does not contain a classical enhancer, and that the enhancement was not due to the splicing reaction per se. Mild DNAse-I digestion of nuclei showed the SPRR2 genes to be tightly associated with the nuclear matrix, in contrast to the other cornified envelope precursor genes mapping to the same chromosomal location (epidermal differentiation complex). In vitro binding studies indicated that both the proximal promoter and the intron of SPRR2A are required for optimal association of this gene with nuclear matrices. Neither nuclear matrix association nor the relative transcriptional enhancement by the intron changed during keratinocyte differentiation. Apparently, the association of the SPRR2A gene with the nuclear matrix results in a general, differentiation-independent enhancement of gene expression.
PMCID: PMC147987  PMID: 9826750
3.  Interdependent transcription control elements regulate the expression of the SPRR2A gene during keratinocyte terminal differentiation. 
Molecular and Cellular Biology  1996;16(10):5365-5374.
Expression of the SPRR2A gene, a member of the small proline-rich family of cornified cell envelope precursor proteins, is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. In this study, we explored the molecular mechanisms underlying this regulation in transiently transfected primary keratinocytes induced to differentiate in vitro. Deletion mapping and site-directed mutagenesis of SPRR2A promoter-chloramphenicol acetyltransferase constructs indicate that four transcription control elements are essential and sufficient for promoter activity. These elements were further characterized by electrophoretic mobility shift and identified as (i) an inverted octamer doublet, bound by the POU domain factor Oct-11 (Skn-1a/i, Epoc-1), (ii) an interferon-stimulated response element recognized by interferon regulatory factors 1 and 2, (iii) an Ets binding site partially overlapping the interferon-stimulated response element, and (iv) a TG box recognized by the Sp1 family of zinc finger transcription factors. Destruction of a single terminal differentiation element is sufficient to completely abolish transcription from the SPRR2A promoter, indicating that these transcription control elements function in concert in an interdependent manner. Apparently, integration of signals transmitted by the above-mentioned transcription factors is necessary and sufficient to promote gene expression during keratinocyte terminal differentiation.
PMCID: PMC231535  PMID: 8816448
4.  Molecular Mechanism of Proinflammatory Cytokine-Mediated Squamous Metaplasia in Human Corneal Epithelial Cells 
Knowledge of the molecular mechanisms involved in immune-mediated ocular surface keratinization is necessary to develop targeted therapies. In this report, the authors identify novel aspects of the signaling pathways that connect the proinflammatory cytokines IL-1β and IFN-γ with the expression of the squamous cell biomarker SPRR1B.
The cornified envelope protein small proline-rich protein 1B (SPRR1B) is a biomarker for squamous metaplasia. Proinflammatory cytokines IL-1β and IFN-γ are potent inducers of ocular surface keratinization and SPRR1B expression. Here the molecular mechanisms controlling SPRR1B gene expression in response to IL-1β and IFN-γ are elucidated.
A 3-kb fragment of the SPRR1B gene 5′-flanking region was amplified from human chromosome 1, sequentially deleted, and cloned into a luciferase vector. Constructs were transiently transfected into human corneal epithelial cells, and activity was assessed in response to IL-1β, IFN-γ, or basal medium. Functional cis-elements responding to IL-1β and IFN-γ were characterized by site-directed mutagenesis and gel mobility shift assay. Effects of mitogen-activated protein kinases p38, ERK, and JNK were assessed using inhibitors and dominant-negative mutants. Results were validated by real-time RT-PCR.
The first 620 bp of the SPRR1B 5′-flanking region regulated constitutive expression and increased promoter activity in response to IL-1β and IFN-γ. Corresponding cis-elements for IL-1β and IFN-γ were bound by cAMP response element binding protein (CREB) and zinc-finger E-box binding homeobox 1 (ZEB1), respectively. Inhibition of p38 abolished the stimulatory effects of IL-1β and IFN-γ on SPRR1B, whereas inhibition of JNK and ERK had no effect. Dominant-negative mutants targeting p38α and p38β2 blocked cytokine-induced SPRR1B promoter activity and mRNA expression.
SPRR1B is upregulated by the proinflammatory cytokines IL-1β and IFN-γ via p38 MAPK-mediated signaling pathways that lead to the activation of transcription factors CREB and ZEB1, respectively. These results identify key intracellular signaling intermediates involved in the pathogenesis of immune-mediated ocular surface squamous metaplasia.
PMCID: PMC2868474  PMID: 20042643
5.  Nrf2 links epidermal barrier function with antioxidant defense 
EMBO Molecular Medicine  2012;4(5):364-379.
The skin provides an efficient permeability barrier and protects from microbial invasion and oxidative stress. Here, we show that these essential functions are linked through the Nrf2 transcription factor. To test the hypothesis that activation of Nrf2 provides skin protection under stress conditions, we determined the consequences of pharmacological or genetic activation of Nrf2 in keratinocytes. Surprisingly, mice with enhanced Nrf2 activity in keratinocytes developed epidermal thickening, hyperkeratosis and inflammation resembling lamellar ichthyosis. This resulted from upregulation of the cornified envelope proteins small proline-rich proteins (Sprr) 2d and 2h and of secretory leukocyte peptidase inhibitor (Slpi), which we identified as novel Nrf2 targets in keratinocytes. Since Sprrs are potent scavengers of reactive oxygen species and since Slpi has antimicrobial activities, their upregulation contributes to Nrf2's protective function. However, it also caused corneocyte fragility and impaired desquamation, followed by alterations in the epidermal lipid barrier, inflammation and overexpression of mitogens that induced keratinocyte hyperproliferation. These results identify an unexpected role of Nrf2 in epidermal barrier function, which needs to be considered for pharmacological use of Nrf2 activators.
PMCID: PMC3403295  PMID: 22383093
barrier function; inflammation; Nrf2; oxidative stress; skin
6.  Essential Role for c-Jun N-Terminal Kinase 2 in Corneal Epithelial Response to Desiccating Stress 
Archives of ophthalmology  2009;127(12):1625-1631.
To investigate the protective effects of c-Jun N-terminal kinase (JNK)–1 and -2 gene knockout (KO) on the corneal epithelial response to desiccating stress.
The C57BL/6, JNK1KO, and JNK2KO mice were subjected to desiccating stress (DS) for 5 days. The effects of DS on the corneal epithelium were evaluated by measuring corneal smoothness and permeability. Expression of matrix metalloproteinases (MMP)–1, MMP-9, and cornified envelope protein precursors (small prolinerich protein [SPRR]–1a, SPRR-2a, and involucrin) in the corneal epithelia was evaluated by immunostaining and real-time polymerase chain reaction. Collagenase and gelatinase activity in corneal sections as measured with in situ fluorescent assays.
The JNK2KO mice had smoother corneal surfaces and less corneal barrier disruption in response to DS than JNK1KO mice and C57BL/6 wild-type control mice. The DS increased levels of MMP-1, MMP-9, SPRR-1a, SPRR-2a, involucrin immunoreactivity, and mRNA transcripts in the corneal epithelium of JNK1KO and C57BL/6 mice, but not in JNK2KO mice. Knockout of JNK2 prevented DS-induced increase in gelatinase and collagenase activity in the cornea.
The JNK2 protein appears to have an essential role in desiccation-induced corneal epithelial disease by stimulating production of MMP-1, MMP-9, and cornified envelope precursors.
Clinical Relevance
The JNK2 protein could be a novel therapeutic target in dry eye disease.
PMCID: PMC3583514  PMID: 20008718
7.  Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics 
PLoS ONE  2012;7(2):e32449.
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.
PMCID: PMC3284570  PMID: 22384255
8.  Skin Cornification Proteins Provide Global Link between ROS Detoxification and Cell Migration during Wound Healing 
PLoS ONE  2010;5(8):e11957.
Wound healing is a complex dynamic process characterised by a uniform flow of events in nearly all types of tissue damage, from a small skin scratch to myocardial infarction. Reactive oxygen species (ROS) are essential during the healing process at multiple stages, ranging from the initial signal that instigates the immune response, to the triggering of intracellular redox-dependent signalling pathways and the defence against invading bacteria. Excessive ROS in the wound milieu nevertheless impedes new tissue formation. Here we identify small proline-rich (SPRR) proteins as essential players in this latter process, as they directly link ROS detoxification with cell migration. A literature-based meta-analysis revealed their up-regulation in various forms of tissue injury, ranging from heart infarction and commensal-induced gut responses to nerve regeneration and burn injury. Apparently, SPRR proteins have a far more widespread role in wound healing and tissue remodelling than their established function in skin cornification. It is inferred that SPRR proteins provide injured tissue with an efficient, finely tuneable antioxidant barrier specifically adapted to the tissue involved and the damage inflicted. Their recognition as novel cell protective proteins combining ROS detoxification with cell migration will provide new venues to study and manage tissue repair and wound healing at a molecular level.
PMCID: PMC2914756  PMID: 20689819
9.  Entrapment of Conjunctival Goblet Cells by Desiccation-Induced Cornification 
The results of this study showed that exposure to desiccating stress stimulates production of cornified envelope precursors and the tissue transglutaminase enzyme that cross-links them via the ocular surface epithelia. These cornifying cells entrap conjunctival goblet cells and block egress of their mucin contents to the ocular surface. This mechanism may contribute to the mucin deficiency that develops in dry eye.
To evaluate the effects of desiccating stress on conjunctival goblet cell density and morphology and the expression of cornified envelope precursors by the ocular surface epithelia.
Experimental dry eye (EDE) was created in C57BL/6 mice. Real-time PCR evaluated the expression of cornified envelope (CE) precursor proteins (involucrin and small proline-rich [Sprr] -1a, -1b, -2a, -2b, -2f, and -2g proteins), the cross-linking transglutaminase 1 enzyme (Tg-1) and Muc5AC mRNA transcripts by the ocular surface epithelia. Laser scanning confocal microscopy evaluated the expression of the CE precursor proteins Tg-1 and Muc5AC in cryosections. Tg-1 activity was measured by a fluorescein cadaverine assay. Muc5AC concentration was measured by ELISA.
Levels of involucrin; Sprr-1a, -1b, -2a, -2b, -2f, and -2g; and Tg1–1 mRNA transcripts in ocular surface tissues increased in response to desiccating stress. Expression and activity of Tg in the conjunctiva markedly increased after EDE. Desiccating stress caused progressive loss of mucin-filled goblet cells. The apical portion of the remaining conjunctival goblet cells became entrapped by adjacent stratified apical epithelia expressing increased levels of cornified envelope precursors.
Exposure to desiccating stress stimulates ocular surface epithelia to produce cornified envelope precursors and the tissue transglutaminase enzyme that cross-links them. This effect is accompanied by loss of mucin-filled goblet cells and entrapment of mucin contents in the remaining ones by cornifying cells that block the egress of mucin contents to the ocular surface. This mechanism may contribute to the conjunctival mucin deficiency that develops in dry eye.
PMCID: PMC3109039  PMID: 21421863
10.  Expression and Regulation of Cornified Envelope Proteins in Human Corneal Epithelium 
Stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery, termed the cornified envelope. The purpose of this study was to evaluate the presence and distribution of cornified envelope precursors in human corneal epithelium, their expression in human corneal epithelial cell cultures, and the effect of ultraviolet radiation (UVB) and transglutaminase (TG) inhibition on their expression.
Tissue distribution of small proline-rich proteins (SPRRs) and filaggrin and involucrin was studied in human cornea sections by immunofluorescence staining. Primary human corneal epithelial cells (HCECs) from limbal explants were used in cell culture experiments. A single dose of UVB at 20 mJ/cm2 was used to stimulate these cells, in the presence or absence of mono-dansyl cadaverine (MDC), a TG inhibitor. SPRR2 and involucrin protein levels were studied by immunofluorescence staining and Western blot analysis. Gene expression of 12 proteins was investigated by semiquantitative reverse transcription–polymerase chain reaction.
In human cornea tissue, SPRR1, SPRR2, filaggrin, and involucrin protein expression were detected in the central and peripheral corneal and limbal epithelium. In HCECs, SPRR2 and involucrin proteins were detected in the cytosolic fraction, and involucrin levels increased after UVB. Both SPRR2 and involucrin levels accumulated in the presence of MDC. Nine genes including involucrin, SPRR (types 1A, 1B, 2A, 2B, and 3), late envelope protein (LEP) 1 and 16, and filaggrin were expressed by HCECs. SPRR 4, loricrin, and LEP 6 transcripts were not detected. UVB downregulated SPRR (2A, 2B) and LEP 1 transcripts.
Various envelope precursors are expressed in human corneal epithelium and in HCECs, acute UVB stress differentially alters their expression in HCECs. The expression of envelope precursors and their rapid modulation by UVB supports the role of these proteins in the regulation of ocular surface stress. TG function may be relevant in the regulation of soluble precursors in UVB-stimulated corneal epithelium.
PMCID: PMC2906387  PMID: 16639001
11.  Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice 
EMBO Molecular Medicine  2014;6(4):442-457.
The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/“metabolizing acquired dioxin-induced skin hamartomas” (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis.
PMCID: PMC3992072  PMID: 24503019
acne; sebaceous gland; Nrf2; oxidative stress; skin
12.  Low humidity environmental challenge causes barrier disruption and cornification of the mouse corneal epithelium in mice via a c-jun N-terminal kinase 2 (JNK2) pathway 
Experimental Eye Research  2011;94(1):150-156.
Patients with tear dysfunction often experience increased irritation symptoms when subjected to drafty and/or low humidity environmental conditions. The purpose of this study was to investigate the effects of low humidity stress (LHS) on corneal barrier function and expression of cornified envelope (CE) precursor proteins in the epithelium of C57BL/6 and c-jun N-terminal kinase 2 (JNK2) knockout (KO) mice. LHS was induced in both strains by exposure to an air draft for 15 (LHS15D) or 30 days (LHS30D) at a relative humidity <30%RH. Nonstressed (NS) mice were used as controls. Oregon-green-dextran uptake was used to measure corneal barrier function. Levels of small proline rich protein (SPRR)-2, involucrin, occludin, and MMP-9 were evaluated by immunofluorescent staining in cornea sections. Wholemount corneas immunostained for occludin were used to measure mean apical cell area. Gelatinase activity was evaluated by in situ zymography. Expression of MMP, CE and inflammatory cytokine genes was evaluated by q-PCR. C57BL/6 mice exposed to LHS15D showed corneal barrier dysfunction, decreased apical corneal epithelial cell area, higher MMP-9 expression and gelatinase activity and increased involucrin and SPRR-2 immunoreactivity in the corneal epithelium compared to NS mice. JNK2KO mice were resistant to LHS-induced corneal barrier disruption. MMP-3,-9,-13, IL-1α, IL-1β, involucrin and SPRR-2a RNA transcripts were significantly increased in C57BL/6 mice at LHS15D, while no change was noted in JNK2KO mice. LHS is capable of altering corneal barrier function, promoting pathologic alteration of the TJ complex and stimulating production of CE proteins by the corneal epithelium. Activation of the JNK2 signaling pathway contributes to corneal epithelial barrier disruption in LHS.
PMCID: PMC3259221  PMID: 22166618
low humidity stress; corneal barrier function; cornified envelop proteins; JNK2 pathway
13.  Small Proline-Rich Protein 1B (SPRR1B) Is a Biomarker for Squamous Metaplasia in Dry Eye Disease 
Squamous metaplasia occurs in ocular surface diseases like Sjögren’s syndrome (SS). It is a phenotypic change whereby epithelial cells initiate synthesis of squamous cell-specific proteins such as small proline-rich protein 1B (SPRR1B) that result in pathologic keratin formation on the ocular surface. The authors hypothesized that inflammation is a key inducer of pathologic keratinization and that SPRR1B represents an analytical biomarker for the study of the molecular mechanisms.
Real-time quantitative RT-PCR and immunohistochemistry were used to examine SPRR1B mRNA and protein in two different mouse models of dry eye and patients with SS. Adoptive transfer of mature lymphocytes from mice lacking the autoimmune regulator (aire) gene was performed to examine the role of inflammation as an inducer of squamous metaplasia. SPRR1B expression in response to several cytokines was examined in vitro, whereas the expression of cytokines IL1β and IFNγ was quantified in ocular tissues of aire-deficient mice and patients with SS.
SPRR1B was increased across the ocular surface of mice with both desiccating stress and autoimmune-mediated, aqueous-deficient dry eye and in patients with SS. Adoptive transfer of CD4+ T cells from aire-deficient mice to immunodeficient recipients caused advanced ocular surface keratinization. IL1α, IL1β, IL6, IFNγ, and TNFα induced SPRR1B expression in vitro and the local expression of IL1β and IFNγ was elevated in ocular tissues of patients with SS and aire-deficient mice.
SPRR1B is a valid biomarker for the study of the molecular mechanisms of squamous metaplasia. There is a definitive link between inflammation and squamous metaplasia in autoimmune-mediated dry eye disease, with IL1β and IFNγ likely acting as key participants.
PMCID: PMC2574421  PMID: 18172072
14.  Differentiation-Associated Genes Regulated by c-Jun and Decreased in the Progression of Esophageal Squamous Cell Carcinoma 
PLoS ONE  2014;9(5):e96610.
Transcription factor c-Jun plays a key role in controlling epithelium cell proliferation, apoptosis and differentiation. However, molecular mechanism and biological functions of c-Jun in squamous differentiation and the progression of esophageal squamous cell carcinoma (ESCC) remain elusive. In this study, we found that c-Jun bound directly to the promoter region, and activated the transcription of differentiation-associated genes including cystatin A, involucrin and SPRR3 in vivo. Ectopic expression of c-Jun enhanced SPRR3 transactivation in KYSE450 cells. Conversely, TAM67, a dominant negative mutant of c-Jun, inhibited SPRR3 transactivation. c-Jun increased expression of SPPR3 mainly via a PKC/JNK pathway in response to TPA in KYSE450 cells. Furthermore, c-Jun was remarkably reduced in esophageal cancer. Interestingly, cystatin A, involucrin and SPRR3 were significantly downregulated as well, and associated with differentiation grade. Expression of c-Jun was correlated with the expression of these genes in normal epithelium and ESCC. Importantly, the expression of these genes was remarkably decreased during the malignant transformation from normal epithelium to low-grade intraepithelial neoplasia (LGIN) or high-grade intraepithelial neoplasia (HGIN). The expression of cystatin A and involucrin was significantly reduced from LGIN to HGIN. These results suggest c-Jun was involved in the regulation of differentiation-associated genes in ESCC. These genes might serve as the potential markers in distinguishing normal epithelium from esophageal squamous intraepithelial neoplasia.
PMCID: PMC4010476  PMID: 24796531
15.  Expression of SPRR3 is associated with tumor cell proliferation and invasion in glioblastoma multiforme 
Oncology Letters  2013;7(2):427-432.
Esophagin, also known as small proline-rich protein 3 (SPRR3), has been demonstrated to be important in the initiation and progression of numerous types of tumor, including colorectal and breast cancer. However, studies concerning the biological functions of SPRR3 in glioblastoma multiforme (GBM) are limited. Therefore, we aimed to identify the functions and molecular mechanisms underlying the role of SPRR3 in GBM. Hypomethylation of SPRR3 was observed and associated with a poor clinical outcome in GBM patients compared with healthy individuals by using gene methylation profiling. The present study was performed to investigate the expression status and effects of SPRR3 in GBM. The U251 cell line was used in the functional analyses. Cell growth was examined by MTT and colony formation assay. Cell invasion was measured using the Transwell invasion assay. The expression of SPRR3 in tissue samples was examined by immunohistochemistry. The results revealed that the overexpression of SPRR3 accelerates U251 cell proliferation and invasion. It was also observed that SPRR3 was markedly upregulated in 72.7% of GBM samples (24/33) compared with the normal tissue. These results suggest that an increased expression of SPRR3 is involved in tumorigenesis.
PMCID: PMC3881942  PMID: 24396461
glioblastoma; SPRR3; cellular proliferation; invasion; methylation profiling
16.  Expression of the regeneration-associated protein SPRR1A in primary sensory neurons and spinal cord of the adult mouse following peripheral and central injury 
Small proline-rich repeat protein 1A (SPRR1A) is expressed in dorsal root ganglion (DRG) neurons following peripheral nerve injury but it is not known whether SPRR1A is differentially expressed following injury to peripheral versus central DRG projections and a detailed characterisation of expression in sensory neuron sub-populations and spinal cord has not been performed. Here we use immunocytochemical techniques to characterise SPRR1A expression following sciatic nerve, dorsal root and dorsal column injury in adult mice. SPRR1A was not detected in naïve spinal cord, DRG or peripheral nerves and there was minimal expression following injury to the centrally projecting branches of DRG neurons. However, following peripheral (sciatic) nerve injury, intense SPRR1A immunoreactivity was observed in the dorsal horn and motoneurons of the spinal cord, in L4/5 DRG neurons and in the injured nerve. A time-course study comparing expression following sciatic nerve crush and transection revealed maximum SPRR1A levels at day 7 in both models. However, while SPRR1A was down-regulated to baseline by 30 days post-lesion following crush injury, it remained elevated 30 days after transection. Cell-size and double-labelling studies revealed that SPRR1A was expressed by DRG cells of all sizes and co-localized with classical markers of DRG subpopulations and their primary afferent terminals. High co-expression of SPRR1A with activating transcription factor-3 and growth-associated protein-43 was observed, indicating that it is expressed by injured and regenerating neurons. This study supports the hypothesis that SPRR1A is a regeneration-associated gene and that SPRR1A provides a valuable marker to assess the regenerative potential of injured neurons.
PMCID: PMC3339437  PMID: 19107756
nerve injury and repair; axonal regeneration; immunolabelling; primary afferents; dorsal root ganglia; regeneration-associated genes
17.  Grainyhead-like 2 (GRHL2) inhibits keratinocyte differentiation through epigenetic mechanism 
Cell Death & Disease  2012;3(12):e450-.
We recently identified Grainyhead-like 2 (GRHL2), a mammalian homolog of Grainyhead in Drosophila, to be a novel transcription factor that regulates hTERT gene expression and enhances proliferation of normal human epidermal keratinocytes (NHEK). In the current study, we show that GRHL2 impairs keratinocyte differentiation through transcriptional inhibition of the genes clustered at the epidermal differentiation complex (EDC), located at chromosome 1q21. Gene expression profiling and subsequent in vitro assays revealed consistent downregulation of EDC genes, for example, IVL, KRT1, FLG, LCEs, and SPRRs, in NHEK expressing exogenous GRHL2. In vivo binding assay by chromatin immunoprecipitation revealed GRHL2 association at the promoter regions of its target genes, many of which belong to EDC. Exogenous GRHL2 expression also inhibited recruitment of histone demethylase Jmjd3 to the EDC gene promoters and enhanced the level of histone 3 Lys 27 trimethylation enrichment at these promoters. Survey of GRHL2 expression in human skin tissues demonstrated enhanced protein and mRNA levels in chronic skin lesions with impaired keratinocyte differentiation, for example, atopic dermatitis and psoriasis, compared with normal epidermis. These data indicate that GRHL2 impairs epidermal differentiation by inhibiting EDC gene expression through epigenetic mechanisms and support its role in the hyperproliferative skin diseases.
PMCID: PMC3542624  PMID: 23254293
epidermal differentiation complex; epigenetic; GRHL2; histone methylation; keratinocyte
18.  JUN regulates early transcriptional responses to axonal injury in retinal ganglion cells 
Experimental eye research  2013;112:106-117.
The AP1 family transcription factor JUN is an important molecule in the neuronal response to injury. In retinal ganglion cells (RGCs), JUN is upregulated soon after axonal injury and disrupting JUN activity delays RGC death. JUN is known to participate in the control of many different injury response pathways in neurons, including pathways controlling cell death and axonal regeneration. The role of JUN in regulating genes involved in cell death, ER stress, and regeneration was tested to determine the overall importance of JUN in regulating RGC response to axonal injury. Genes from each of these pathways were transcriptionally controlled following axonal injury and Jun deficiency altered the expression of many of these genes. The differentially expressed genes included, Atf3, Ddit3, Ecel1, Gadd45α, Gal, Hrk, Pten, Socs3, and Sprr1a. Two of these genes, Hrk and Atf3, were tested for importance in RGC death using null alleles of each gene. Disruption of the prodeath Bcl2 family member Hrk did not affect the rate or amount of RGC death after axonal trauma. Deficiency in the ATF/CREB family transcription factor Atf3 did lessen the amount of RGC death after injury, though it did not provide long term protection to RGCs. Since JUN’s dimerization partner determines its transcriptional targets, the expression of several candidate AP1 family members were examined. Multiple AP1 family members were induced by axonal injury and had a different expression profile in Jun deficient retinas compared to wildtype retinas (Fosl1, Fosl2 and Jund). Overall, JUN appears to play a multifaceted role in regulating RGC response to axonal injury.
PMCID: PMC3700614  PMID: 23648575
glaucoma; cell death; regeneration; ER stress; trauma; axonal injury; AP1
19.  Upregulation of SPRR3 Promotes Colorectal Tumorigenesis 
Molecular Medicine  2010;16(7-8):271-277.
Hereditary colorectal cancer develops through a series of well-defined genetic and histological changes. However, elucidation of the canonical pathway based on hereditary colorectal cancer has not provided a clear explanation of the molecular mechanisms of sporadic colorectal cancer. To identify the alterative pathways involved in sporadic colorectal tumorigenesis, we performed gene expression analysis in patients with sporadic colorectal tumors. A comparison analysis of gene expression profiles revealed a pattern of upregulation of small proline rich repeat protein 3 (SPRR3) in tumor samples. SPRR3 has previously been reported to be downregulated in esophageal cancer. However, in the present study, we observed that SPRR3 was strongly upregulated in 31 of 35 samples of sporadic colorectal tumors (88%). We also determined that overexpression of SPRR3 not only accelerates colorectal cancer cell proliferation but also is associated with lymphovascular invasion in colorectal cancer. Moreover, AKT was activated and p53 levels were decreased in cells that overexpressed SPRR3. In contrast to the pattern seen in esophageal cancer, these results suggest that increased expression of SPRR3 is involved in colorectal tumorigenesis.
PMCID: PMC2896463  PMID: 20379613
20.  Nrf2 Transcription Factor, a Novel Target of Keratinocyte Growth Factor Action Which Regulates Gene Expression and Inflammation in the Healing Skin Wound 
Molecular and Cellular Biology  2002;22(15):5492-5505.
Keratinocyte growth factor (KGF) is a potent mitogen for epithelial cells, and it promotes survival of these cells under stress conditions. In a search for KGF-regulated genes in keratinocytes, we identified the gene encoding the transcription factor NF-E2-related factor 2 (Nrf2). Nrf2 is a key player in the cellular stress response. This might be of particular importance during wound healing, where large amounts of reactive oxygen species are produced as a defense against invading bacteria. Therefore, we studied the wound repair process in Nrf2 knockout mice. Interestingly, the expression of various key players involved in wound healing was significantly reduced in early wounds of the Nrf2 knockout animals, and the late phase of repair was characterized by prolonged inflammation. However, these differences in gene expression were not reflected by obvious histological abnormalities. The normal healing rate appears to be at least partially due to an up-regulation of the related transcription factor Nrf3, which was also identified as a target of KGF and which was coexpressed with Nrf2 in the healing skin wound. Taken together, our results reveal novel roles of the KGF-regulated transcription factors Nrf2 and possibly Nrf3 in the control of gene expression and inflammation during cutaneous wound repair.
PMCID: PMC133949  PMID: 12101242
21.  Phosphorylation of Calcineurin at a Novel Serine-Proline Rich Region Orchestrates Hyphal Growth and Virulence in Aspergillus fumigatus 
PLoS Pathogens  2013;9(8):e1003564.
The fungus Aspergillus fumigatus is a leading infectious killer in immunocompromised patients. Calcineurin, a calmodulin (CaM)-dependent protein phosphatase comprised of calcineurin A (CnaA) and calcineurin B (CnaB) subunits, localizes at the hyphal tips and septa to direct A. fumigatus invasion and virulence. Here we identified a novel serine-proline rich region (SPRR) located between two conserved CnaA domains, the CnaB-binding helix and the CaM-binding domain, that is evolutionarily conserved and unique to filamentous fungi and also completely absent in human calcineurin. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of A. fumigatus CnaA in vivo at four clustered serine residues (S406, S408, S410 and S413) in the SPRR. Mutation of the SPRR serine residues to block phosphorylation led to significant hyphal growth and virulence defects, indicating the requirement of calcineurin phosphorylation at the SPRR for its activity and function. Complementation analyses of the A. fumigatus ΔcnaA strain with cnaA homologs from the pathogenic basidiomycete Cryptococcus neoformans, the pathogenic zygomycete Mucor circinelloides, the closely related filamentous fungi Neurospora crassa, and the plant pathogen Magnaporthe grisea, revealed filamentous fungal-specific phosphorylation of CnaA in the SPRR and SPRR homology-dependent restoration of hyphal growth. Surprisingly, circular dichroism studies revealed that, despite proximity to the CaM-binding domain of CnaA, phosphorylation of the SPRR does not alter protein folding following CaM binding. Furthermore, mutational analyses in the catalytic domain, CnaB-binding helix, and the CaM-binding domains revealed that while the conserved PxIxIT substrate binding motif in CnaA is indispensable for septal localization, CaM is required for its function at the hyphal septum but not for septal localization. We defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi in a region absent in humans. These findings suggest the possibility of harnessing this unique SPRR for innovative antifungal drug design to combat invasive aspergillosis.
Author Summary
Invasive fungal infections are a leading cause of death in immunocompromised patients. Translating molecular understanding into tangible clinical benefit has been difficult due to the fact that fungal pathogens and their hosts have similar physiology. The calcineurin pathway is an important signaling cascade in all eukaryotes, and calcineurin inhibitors are powerful immunosuppressants that have revolutionized medicine. Through both genetic and pharmacologic inhibition, we have established that calcineurin is vital for invasive fungal disease. Although the currently available calcineurin inhibitors are active in vitro against the major invasive fungal pathogens, they are also immunosuppressive in the host, limiting therapeutic effectiveness. Here we defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi that is responsible for virulence in the opportunistic human pathogen, Aspergillus fumigatus. This phosphorylation occurs on a cluster of four serine residues located in a unique serine-proline rich domain of calcineurin that is absent in humans. This finding of a new fungal-specific mechanism controlling hyphal growth and virulence represents a new potential target for antifungal drug therapy.
PMCID: PMC3749960  PMID: 23990785
22.  Genetic variation in small proline rich protein 2B as a predictor for asthma among children with eczema 
Small proline rich protein 2B (SPRR2B) is a skin and lung epithelial protein associated with allergic inflammation in mice that has not been evaluated in human atopic diseases.
To determine whether single-nucleotide polymorphisms (SNPs) in SPRR2B are associated with childhood eczema and with the phenotype of childhood eczema combined with asthma.
Genotyping for SPRR2B and filaggrin (FLG) was performed in 2 independent populations: the Cincinnati Childhood Allergy & Air Pollution Study (CCAAPS; N = 762; birth-age, 4 years) and the Greater Cincinnati Pediatric Clinical Repository (GCPCR;N = 1152; ages 5–10 years). Eczema and eczema plus asthma were clinical outcomes based on parental report and clinician’s diagnosis. Genetic analyses were restricted to whites and adjusted for sex in both cohorts and adjusted for environmental covariates in CCAAPS.
Variants in SPRR2B were not significantly associated with eczema in either cohort after Bonferroni adjustment. Children from both cohorts with the CC genotype of the SPRR2B rs6693927 SNP were at 4 times the risk for eczema plus asthma (adjusted odds ratio, 4.1; 95% confidence interval, 1.5– 10.9; P = .005 in CCAAPS; and adjusted odds ratio, 4.0; 95% confidence interval, 1.8 –9.1; P <.001 in the GCPCR), however. SNPs in SPRR2B were not in strong linkage disequilibrium with the R501X and del2282 FLG mutations, and these findings were independent of FLG.
An SNP in SPRR2B was predictive of asthma among white children with eczema from 2 independent populations. SPRR2B polymorphisms may serve as important predictive markers for the combined eczema plus asthma phenotype.
PMCID: PMC3759990  PMID: 22374195
23.  A comparison of epithelial-to-mesenchymal transition and re-epithelialization 
Seminars in cancer biology  2012;22(5-6):471-483.
Wound healing and cancer metastasis share a common starting point, namely, a change in the phenotype of some cells from stationary to motile. The term, epithelial-to-mesenchymal transition (EMT) describes the changes in molecular biology and cellular physiology that allow a cell to transition from a sedentary cell to a motile cell, a process that is relevant not only for cancer and regeneration, but also for normal development of multicellular organisms. The present review compares the similarities and differences in cellular response at the molecular level as tumor cells enter EMT or as keratinocytes begin the process of re-epithelialization of a wound. Looking toward clinical interventions that might modulate these processes, the mechanisms and outcomes of current and potential therapies are reviewed for both anti-cancer and pro-wound healing treatments related to the pathways that are central to EMT. Taken together, the comparison of re-epithelialization and tumor EMT serves as a starting point for the development of therapies that can selectively modulate different forms of EMT.
PMCID: PMC3595494  PMID: 22863788
Epithelial-to-mesenchymal transition; Keratinocyte; Dermal wound healing; Metastasis; Metastasis
24.  Hole-in-One Mutant Phenotypes Link EGFR/ERK Signaling to Epithelial Tissue Repair in Drosophila 
PLoS ONE  2011;6(11):e28349.
Epithelia act as physical barriers protecting living organisms and their organs from the surrounding environment. Simple epithelial tissues have the capacity to efficiently repair wounds through a resealing mechanism. The known molecular mechanisms underlying this process appear to be conserved in both vertebrates and invertebrates, namely the involvement of the transcription factors Grainy head (Grh) and Fos. In Drosophila, Grh and Fos lead to the activation of wound response genes required for epithelial repair. ERK is upstream of this pathway and known to be one of the first kinases to be activated upon wounding. However, it is still unclear how ERK activation contributes to a proper wound response and which molecular mechanisms regulate its activation.
Methodology/Principal Findings
In a previous screen, we isolated mutants with defects in wound healing. Here, we describe the role of one of these genes, hole-in-one (holn1), in the wound healing process. Holn1 is a GYF domain containing protein that we found to be required for the activation of several Grh and Fos regulated wound response genes at the wound site. We also provide evidence suggesting that Holn1 may be involved in the Ras/ERK signaling pathway, by acting downstream of ERK. Finally, we show that wound healing requires the function of EGFR and ERK signaling.
Based on these data, we conclude that holn1 is a novel gene required for a proper wound healing response. We further propose and discuss a model whereby Holn1 acts downstream of EGFR and ERK signaling in the Grh/Fos mediated wound closure pathway.
PMCID: PMC3226689  PMID: 22140578
25.  Epithelial-Mesenchymal Transition Induced by Senescent Fibroblasts 
Cancer Microenvironment  2011;5(1):39-44.
Depending on the cell type and tissue environment, epithelial and mesenchymal cell phenotypes are not static and can be highly dynamic. Epithelial-mesenchymal transitions (EMTs) and reverse EMTs provide flexibility during embryogenesis. While EMTs are a critical normal process during development and wound healing, properties of the EMT have been implicated in human pathology, particularly cancer metastasis. A normal undamaged epithelium does not typically exhibit features of an EMT. However, particularly under the influence of the surrounding microenvironment, cancer cells may reactivate developmental phenotypes out of context in the adult. This reactivation, such as the EMT, can facilitate tumor cell invasion and metastasis, and therefore is a major mechanism of tumor progression. Conversely, cellular senescence, which is associated with aging, is a process by which cells enter a state of permanent cell cycle arrest, thereby constituting a potent tumor suppressive mechanism. However, accumulating evidence shows that senescent cells can have deleterious effects on the tissue microenvironment. The most significant of these effects is the acquisition of a senescence-associated secretory phenotype (SASP) that turns senescent fibroblasts into pro-inflammatory cells having the ability to promote tumor progression, in part by inducing an EMT in nearby epithelial cells. Here, we summarize the potential impacts of SASP factors, particularly interleukins, on tissue microenvironments and their ability to stimulate tumor progression through induction of an EMT.
PMCID: PMC3343197  PMID: 21706180
Aging; Senescence; Cytokines; Interleukins; Proliferation; Invasion; Migration

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