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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.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  Drosophila Histone Deacetylase-3 Controls Imaginal Disc Size through Suppression of Apoptosis 
PLoS Genetics  2008;4(2):e1000009.
Histone deacetylases (HDACs) execute biological regulation through post-translational modification of chromatin and other cellular substrates. In humans, there are eleven HDACs, organized into three distinct subfamilies. This large number of HDACs raises questions about functional overlap and division of labor among paralogs. In vivo roles are simpler to address in Drosophila, where there are only five HDAC family members and only two are implicated in transcriptional control. Of these two, HDAC1 has been characterized genetically, but its most closely related paralog, HDAC3, has not. Here we describe the isolation and phenotypic characterization of hdac3 mutations. We find that both hdac3 and hdac1 mutations are dominant suppressors of position effect variegation, suggesting functional overlap in heterochromatin regulation. However, all five hdac3 loss-of-function alleles are recessive lethal during larval/pupal stages, indicating that HDAC3 is essential on its own for Drosophila development. The mutant larvae display small imaginal discs, which result from abnormally elevated levels of apoptosis. This cell death occurs as a cell-autonomous response to HDAC3 loss and is accompanied by increased expression of the pro-apoptotic gene, hid. In contrast, although HDAC1 mutants also display small imaginal discs, this appears to result from reduced proliferation rather than from elevated apoptosis. The connection between HDAC loss and apoptosis is important since HDAC inhibitors show anticancer activities in animal models through mechanisms involving apoptotic induction. However, the specific HDACs implicated in tumor cell killing have not been identified. Our results indicate that protein deacetylation by HDAC3 plays a key role in suppression of apoptosis in Drosophila imaginal tissue.
Author Summary
Histone deacetylases (HDACs) are enzymes that reverse acetylation of protein substrates inside the cell. Like phosphorylation, acetylation/deacetylation can alter the biochemical properties of a protein target and thereby regulate its functions. Histones are a major target of certain HDACs. When histones become deacetylated, the biochemical properties of the local chromatin are altered, which can contribute to gene silencing. HDACs can also act upon protein substrates besides histones. There are multiple HDACs encoded in animal genomes, with eleven HDACs in humans. Thus, it becomes complicated to determine which individual HDACs exert which biological functions in vivo. To address this, we have isolated and studied mutations that specifically disrupt a single HDAC, HDAC3, in Drosophila. We find that a major function of HDAC3 is to prevent programmed cell death from occurring abnormally in certain fly tissues. This finding has implications for anticancer strategies since HDAC chemical inhibitors can reduce tumors in animal models through induction of cell death. Our study identifies HDAC3 as a single HDAC among many that can play a key role in control of cell death and suggests that this version of the enzyme should be further investigated for regulatory roles in tumor cell killing versus survival.
PMCID: PMC2265479  PMID: 18454196
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 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
11.  HIPK2 modulates p53 activity towards pro-apoptotic transcription 
Molecular Cancer  2009;8:85.
Activation of p53-mediated gene transcription is a critical cellular response to DNA damage and involves a phosphorylation-acetylation cascade of p53. The discovery of differences in the response to different agents raises the question whether some of the p53 oncosuppressor functions might be exerted by different posttranslational modifications. Stress-induced homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates p53 at serine-46 (Ser46) for p53 apoptotic activity; p53 acetylation at different C-terminus lysines including p300-mediated lysine-382 (Lys382) is also required for full activation of p53 transcriptional activity. The purpose of the current study was to evaluate the interplay among HIPK2, p300, and p53 in p53 acetylation and apoptotic transcriptional activity in response to drug by using siRNA interference, p300 overexpression or deacetylase inhibitors, in cancer cells.
Knockdown of HIPK2 inhibited both adriamycin-induced Ser46 phosphorylation and Lys382 acetylation in p53 protein; however, while combination of ADR and zinc restored Ser46 phosphorylation it did not recover Lys382 acetylation. Chromatin immunoprecipitation studies showed that HIPK2 was required in vivo for efficient p300/p53 co-recruitment onto apoptotic promoters and that both p53 modifications at Ser46 and Lys382 were necessary for p53 apoptotic transcription. Thus, p53Lys382 acetylation in HIPK2 knockdown as well as p53 apoptotic activity in response to drug could be rescued by p300 overexpression. Similar effect was obtained with the Sirt1-inhibitor nicotinamide. Interestingly trichostatin A (TSA), the inhibitor of histone deacetylase complexes (HDAC) did not have effect, suggesting that Sirt1 was the deacetylase involved in p53 deacetylation in HIPK2 knockdown.
These results reveal a novel role for HIPK2 in activating p53 apoptotic transcription. Our results indicate that HIPK2 may regulate the balance between p53 acetylation and deacetylation, by stimulating on one hand co-recruitment of p300 and p53Lys382 on apoptotic promoters and on the other hand by inhibiting Sirt1 deacetylase activity. We attempted to reactivate p53 apoptotic transcriptional activity by rescuing both Ser46 and Lys382 modification in response to drug. Our data propose combination strategies for the treatment of tumors with dysfunctional p53 and/or HIPK2 that include classical chemotherapy with pharmacological or natural agents such as Sirt1-deacetylase inhibitors or zinc, respectively.
PMCID: PMC2768676  PMID: 19828042
12.  Isolation and characterization of a novel epithelium-specific transcription factor, ESE-1, a member of the ets family. 
Molecular and Cellular Biology  1997;17(8):4419-4433.
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.
PMCID: PMC232296  PMID: 9234700
13.  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
14.  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
15.  Histone Deacetylase 3 Interacts with and Deacetylates Myocyte Enhancer Factor 2▿ †  
Molecular and Cellular Biology  2006;27(4):1280-1295.
The myocyte enhancer factor 2 (MEF2) family of transcription factors is not only important for controlling gene expression in normal cellular programs, like muscle differentiation, T-cell apoptosis, neuronal survival, and synaptic differentiation, but has also been linked to cardiac hypertrophy and other pathological conditions. Lysine acetylation has been shown to modulate MEF2 function, but it is not so clear which deacetylase(s) is involved. We report here that treatment of HEK293 cells with trichostatin A or nicotinamide upregulated MEF2D acetylation, suggesting that different deacetylases catalyze the deacetylation. Related to the trichostatin A sensitivity, histone deacetylase 4 (HDAC4) and HDAC5, two known partners of MEF2, exhibited little deacetylase activity towards MEF2D. In contrast, HDAC3 efficiently deacetylated MEF2D in vitro and in vivo. This was specific, since HDAC1, HDAC2, and HDAC8 failed to do so. While HDAC4, HDAC5, HDAC7, and HDAC9 are known to recognize primarily the MEF2-specific domain, we found that HDAC3 interacts directly with the MADS box. In addition, HDAC3 associated with the acetyltransferases p300 and p300/CBP-associated factor (PCAF) to reverse autoacetylation. Furthermore, the nuclear receptor corepressor SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) stimulated the deacetylase activity of HDAC3 towards MEF2 and PCAF. Supporting the physical interaction and deacetylase activity, HDAC3 repressed MEF2-dependent transcription and inhibited myogenesis. These results reveal an unexpected role for HDAC3 and suggest a novel pathway through which MEF2 activity is controlled in vivo.
PMCID: PMC1800729  PMID: 17158926
16.  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
17.  RASSF1A Suppresses Cell Migration through Inactivation of HDAC6 and Increase of Acetylated α-Tubulin 
The RAS association domain family protein 1 (RASSF1) has been implicated in a tumor-suppressive function through the induction of acetylated α-tubulin and modulation of cell migration. However, the mechanisms of how RASSF1A is associated with acetylation of α-tubulin for controlling cell migration have not yet been elucidated. In this study, we found that RASSF1A regulated cell migration through the regulation of histon deacetylase 6 (HDAC6), which functions as a tubulin deacetylase.
Materials and Methods
The cell migration was assessed using wound-healing and transwell assays. The role of RASSF1A on cell migration was examined by immunofluorescence staining, HDAC activity assay and western blot analysis.
Cell migration was inhibited and cell morphology was changed in RASSF1A-transfected H1299 cells, compared with controls, whereas HDAC6 protein expression was not changed by RASSF1A transfection in these cells. However, RASSF1A inhibited deacetylating activity of HDAC6 protein and induced acetylated α-tubulin expression. Furthermore, acetylated α-tubulin and HDAC6 protein were co-localized in the cytoplasm in RASSF1A-transfected H1299 cells. Conversely, when the endogenous RASSF1A expression in HeLa cells was blocked with RASSF1A siRNA treatment, acetylated α-tubulin was co-localized with HDAC6 protein throughout the whole cells, including the nucleus, compared with scramble siRNA-treated HeLa cells. The restoration of RASSF1A by 5-Aza-dC treatment also induced acetylated α-tubulin through inhibition of HDAC6 activity that finally resulted in suppressing cell migration in H1299 cells. To further confirm the role of HDAC6 in RASSF1A-mediated cell migration, the HDAC6 expression in H1299 cells was suppressed by using HDAC6 siRNA, and cell motility was found to be decreased through enhanced acetylated α-tubulin.
The results of this study suggest that the inactivation of HDAC6 by RASSF1A regulates cell migration through increased acetylated α-tubulin protein.
PMCID: PMC3710963  PMID: 23864847
Cell movement; HDAC6; Lung neoplasms; RASSF1A; Tumor suppressor genes
18.  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
19.  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
20.  Neurotrophins and Nerve Regeneration-associated genes are expressed in the Cornea after Lamellar Flap Surgery 
Cornea  2012;31(12):1460-1467.
To determine the in vivo expression of neurotrophins (NTs) and nerve regeneration-associated genes (RAGs) after surgically creating a hinged lamellar corneal flap in thy1-YFP mice.
Lamellar corneal flaps with multiple hinges were created in thy1-YFP mice. Mice were sacrificed weeks 2, 4, and 8. Quantitative PCR was performed to determine the expression of NTs and RAGs in the corneas following lamellar transection. Nerve growth factor (Ngf), Brain-derived neurotrophic factor (Bdnf), Glial cell-derived neurotrophic factor (Gdnf), Neurotrophin-3 (Ntf3), Neurotrophin 5 (Ntf5), Small proline-rich repeat protein 1A (Sprr1a), Growth-associated protein 43 (Gap43) and Beta III tubulin (Tubb3) gene expressions were analyzed. Whole-mount confocal immunofluorescence and Western analyses were performed for localization and abundance of robustly expressed genes.
Sprouts of fine YFP positive fronds emanating from transected (injured) nerve bundles were seen in the flap area at 2 weeks onwards. Bdnf and Sprr1a were robustly and significantly expressed at 2 weeks postoperatively (> 2 folds increase in expression and p < 0.05). Bdnf localized to thy1-YFP+ cells in operated corneas. Sprr1a localized to corneal epithelial cell membranes. At 8 weeks, none of the NTs and RAGs had increased expression. Bdnf (ρ = 0.73, p = 0.001) and Sprr1a (ρ = 0.76, p = 0.001) showed a significant positive correlation with Tubb3.
The neurotrophin Bdnf and regeneration-associated gene Sprr1a are robustly and significantly expressed during corneal nerve regeneration in vivo.
PMCID: PMC3612527  PMID: 22673847
Regeneration-associated gene; neurotrophin; corneal nerve; nerve regeneration; lamellar flap
21.  Histone acetylation mediates epigenetic regulation of transcriptional reprogramming in insects during metamorphosis, wounding and infection 
Frontiers in Zoology  2012;9:25.
Gene expression in eukaryotes is regulated by histone acetylation/deacetylation, an epigenetic process mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) whose opposing activities are tightly regulated. The acetylation of histones by HATs increases DNA accessibility and promotes gene expression, whereas the removal of acetyl groups by HDACs has the opposite effect.
We explored the role of HDACs and HATs in epigenetic reprogramming during metamorphosis, wounding and infection in the lepidopteran model host Galleria mellonella. We measured the expression of genes encoding components of HATs and HDACs to monitor the transcriptional activity of each enzyme complex and found that both enzymes were upregulated during pupation. Specific HAT inhibitors were able to postpone pupation and to reduce insect survival following wounding, whereas HDAC inhibitors accelerated pupation and increased survival. The administration of HDAC inhibitors modulated the expression of effector genes with key roles in tissue remodeling (matrix metalloproteinase), the regulation of sepsis (inhibitor of metalloproteinases from insects) and host defense (antimicrobial peptides), and simultaneously induced HAT activity, suggesting that histone acetylation is regulated by a feedback mechanism. We also discovered that both the entomopathogenic fungus Metarhizium anisopliae and the human bacterial pathogen Listeria monocytogenes can delay metamorphosis in G. mellonella by skewing the HDAC/HAT balance.
Our study provides for the first evidence that pathogenic bacteria can interfere with the regulation of HDACs and HATs in insects which appear to manipulate host immunity and development. We conclude that histone acetylation/deacetylation in insects mediates transcriptional reprogramming during metamorphosis and in response to wounding and infection.
PMCID: PMC3538701  PMID: 23035888
Epigenetics; Histone acetylation; Development; Metamorphosis; Immunity; Galleria mellonella
22.  Regulation of Transcription Factor YY1 by Acetylation and Deacetylation 
Molecular and Cellular Biology  2001;21(17):5979-5991.
YY1 is a sequence-specific DNA-binding transcription factor that has many important biological roles. It activates or represses many genes during cell growth and differentiation and is also required for the normal development of mammalian embryos. Previous studies have established that YY1 interacts with histone acetyltransferases p300 and CREB-binding protein (CBP) and histone deacetylase 1 (HDAC1), HDAC2, and HDAC3. Here, we present evidence that the activity of YY1 is regulated through acetylation by p300 and PCAF and through deacetylation by HDACs. YY1 was acetylated in two regions: both p300 and PCAF acetylated the central glycine-lysine-rich domain of residues 170 to 200, and PCAF also acetylated YY1 at the C-terminal DNA-binding zinc finger domain. Acetylation of the central region was required for the full transcriptional repressor activity of YY1 and targeted YY1 for active deacetylation by HDACs. However, the C-terminal region of YY1 could not be deacetylated. Rather, the acetylated C-terminal region interacted with HDACs, which resulted in stable HDAC activity associated with the YY1 protein. Finally, acetylation of the C-terminal zinc finger domain decreased the DNA-binding activity of YY1. Our findings suggest that in the natural context, YY1 activity is regulated through intricate mechanisms involving negative feedback loops, histone deacetylation, and recognition of the cognate DNA sequence affected by acetylation and deacetylation of the YY1 protein.
PMCID: PMC87316  PMID: 11486036
23.  Epigenetics and Chromatin Remodeling Play a Role in Lung Disease 
Tanaffos  2011;10(4):7-16.
Epigenetics is defined as heritable changes that affect gene expression without altering the DNA sequence. Epigenetic regulation of gene expression is facilitated through different mechanisms such as DNA methylation, histone modifications and RNA-associated silencing by small non-coding RNAs. All these mechanisms are crucial for normal development, differentiation and tissue-specific gene expression. These three systems interact and stabilize one another and can initiate and sustain epigenetic silencing, thus determining heritable changes in gene expression. Histone acetylation regulates diverse cellular functions including inflammatory gene expression, DNA repair and cell proliferation. Transcriptional coactivators possess intrinsic histone acetyltransferase activity and this activity drives inflammatory gene expression. Eleven classical histone deacetylases (HDACs) act to regulate the expression of distinct subsets of inflammatory/immune genes. Thus, loss of HDAC activity or the presence of HDAC inhibitors can further enhance inflammatory gene expression by producing a gene-specific change in HAT activity. For example, HDAC2 expression and activity are reduced in lung macrophages, biopsy specimens, and blood cells from patients with severe asthma and smoking asthmatics, as well as in patients with chronic obstructive pulmonary disease (COPD). This may account, at least in part, for the enhanced inflammation and reduced steroid responsiveness seen in these patients. Other proteins, particularly transcription factors, are also acetylated and are targets for deacetylation by HDACs and sirtuins, a related family of 7 predominantly protein deacetylases. Thus the acetylation/deacetylation status of NF-κB and the glucocorticoid receptor can also affect the overall expression pattern of inflammatory genes and regulate the inflammatory response. Understanding and targeting specific enzymes involved in this process might lead to new therapeutic agents, particularly in situations in which current anti-inflammatory therapies are suboptimal.
PMCID: PMC4153170  PMID: 25191382
HDAC; Inflammatory cells; COPD; Asthma
24.  Histone Deacetylase 2 (HDAC2) Regulates Chromosome Segregation and Kinetochore Function via H4K16 Deacetylation during Oocyte Maturation in Mouse 
PLoS Genetics  2013;9(3):e1003377.
Changes in histone acetylation occur during oocyte development and maturation, but the role of specific histone deacetylases in these processes is poorly defined. We report here that mice harboring Hdac1−/+/Hdac2−/− or Hdac2−/− oocytes are infertile or sub-fertile, respectively. Depleting maternal HDAC2 results in hyperacetylation of H4K16 as determined by immunocytochemistry—normal deacetylation of other lysine residues of histone H3 or H4 is observed—and defective chromosome condensation and segregation during oocyte maturation occurs in a sub-population of oocytes. The resulting increased incidence of aneuploidy likely accounts for the observed sub-fertility of mice harboring Hdac2−/− oocytes. The infertility of mice harboring Hdac1−/+/Hdac2−/−oocytes is attributed to failure of those few eggs that properly mature to metaphase II to initiate DNA replication following fertilization. The increased amount of acetylated H4K16 likely impairs kinetochore function in oocytes lacking HDAC2 because kinetochores in mutant oocytes are less able to form cold-stable microtubule attachments and less CENP-A is located at the centromere. These results implicate HDAC2 as the major HDAC that regulates global histone acetylation during oocyte development and, furthermore, suggest HDAC2 is largely responsible for the deacetylation of H4K16 during maturation. In addition, the results provide additional support that histone deacetylation that occurs during oocyte maturation is critical for proper chromosome segregation.
Author Summary
Oocyte development is becoming of increasing interest not only in the broad research community but also within the general public due, in part, to the ever increasing demand for and use of assisted reproductive technologies (ART) to treat human infertility, and because the oocyte-to-embryo transition encompasses a natural reprogramming of gene expression, a process central to forming iPS cells. Dramatic changes in chromatin structure and gene expression occur during oocyte development, but the role of such changes in generating oocytes that are capable of maturing, being fertilized, and giving rise to offspring is very poorly understood. Histone deacetylases (HDACs) are critically involved in modulating chromatin structure. Here, we describe the effect of specifically deleting the gene encoding Hdac2 in mouse oocytes and find the fertility of female mice harboring such oocytes is compromised. Although such mutant oocytes can grow they fail to mature properly to become an egg. The primary defect is that histone H4 acetylated on lysine 16 fails to become deacetylated as the oocyte matures to become an egg, with the consequence that the ability of chromosomes to interact with spindle microtubules is compromised, which in turn leads to improper chromosome segregation.
PMCID: PMC3597510  PMID: 23516383
25.  Immune profile of squamous metaplasia development in autoimmune regulator-deficient dry eye 
Molecular Vision  2009;15:563-576.
Squamous metaplasia of the ocular surface epithelium in severe Sjögren syndrome (SS) dry eye has been implicated to be associated with chronic engagement of immune-mediated inflammation. While the detailed immunopathological mechanism underlying keratinization of the ocular muco-epithelium in this setting remains unclear, mice deficient in the autoimmune regulator gene (Aire) demonstrate SS-like pathological changes in the exocrine organs and ocular surface including squamous metaplasia. Using this murine model, we sought to determine the specific immune events that predict squamous metaplasia of the cornea in Aire deficiency.
Lissamine green staining, goblet cell density, and corneal small proline-rich protein 1B (SPRR1B) were compared in Aire-sufficient and -deficient mice at 4, 8, and 16 weeks of age. Corneal, limbal and conjunctival infiltration of CD4+ and CD8+ T cells as well as CD11c+ and MHC class II (I-Ad+) dendritic cells (DCs) were examined at the same time points. Ordinary least squares regression was used to model SPRR1B’s relationship with lissamine green staining, goblet cell density, and immune cell infiltration.
Lissamine green staining was present in Aire-deficient mice by four weeks of age and increased over time. Compared to Aire-sufficient controls, conjunctival goblet cell density (GCD) decreased and corneal SPRR1B increased in Aire-deficient mice with significant differences noted at both 8 and 16 weeks. Immune-mediated CD4+ T cell infiltration of the conjunctiva and limbus peaked at eight weeks and then decreased. In contrast, corneal T cell infiltration continued to increase over time, reaching a maximum cell number at 16 weeks. CD11c+ myeloid-derived DCs were found in the conjunctiva and limbus at all time points. As the mice aged, there was a notable increase in corneal CD11c+ cell counts. Interestingly, the dynamic of activated MHC class II+ DCs was nearly identical to that of CD4+ T cells, peaking first in the limbus at eight weeks with maximum infiltration of the cornea by 16 weeks. Regression analysis showed that squamous metaplasia biomarker, SPRR1B, is strongly related to the lissamine green staining of the ocular surface. Corneal infiltration of activated DCs was most prognostic of corneal SPRR1B expression while the presence of precursor DCs, activated DCs, and CD4+ T cells in the limbus were also significant predictors of SPRR1B.
Aire-deficient mice represent a useful model to study Sjögren-like autoimmune-mediated ocular surface disease. Results of the current study suggest that squamous cell precursor protein, SPRR1B, provides an important readout to evaluate ocular surface damage and specific events related to immune-mediated inflammation. Results also define an appropriate time frame for interventional studies to develop more effective therapies for keratinizing ocular surface disease.
PMCID: PMC2660375  PMID: 19365590

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