RESUMO
Charcot-Marie-Tooth (CMT) disease is a peripheral neuropathy associated with gene duplication and point mutations in the peripheral myelin protein 22 (PMP22) gene. However, the role of PMP22 in Schwann cell physiology and the mechanisms by which PMP22 mutations cause CMT are not well-understood. On the basis of homology between PMP22 and proteins associated with modulation of ion channels, we hypothesized that PMP22 alters ion channel activity. Using whole-cell electrophysiology, we show here that heterologous PMP22 expression increases the amplitude of currents similar to those ascribed to store-operated calcium (SOC) channels, particularly those involving transient receptor canonical channel 1 (TrpC1). These channels help replenish Ca2+ in the endoplasmic reticulum (ER) following stimulus-induced depletion. Currents with similar properties were recorded in WT but not pmp22-/- mouse Schwann cells. Heterologous expression of the CMT-associated PMP22_L16P variant, which fails to reach the plasma membrane and localizes to the ER, led to larger currents than WT PMP22. Similarly, Schwann cells isolated from Trembler J (TrJ; PMP22_L16P) mice had larger currents than WT littermates. Calcium imaging in live nerves and cultured Schwann cells revealed elevated intracellular Ca2+ in TrJ mice compared with WT. Moreover, we found that PMP22 co-immunoprecipitated with stromal interaction molecule 1 (STIM1), the Ca2+ sensor SOC channel subunit in the ER. These results suggest that in the ER, PMP22 interacts with STIM1 and increases Ca2+ influx through SOC channels. Excess or mutant PMP22 in the ER may elevate intracellular Ca2+ levels, which could contribute to CMT pathology.
Assuntos
Canais de Cálcio/metabolismo , Proteínas da Mielina/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/química , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Retículo Endoplasmático/metabolismo , Potenciais Evocados/efeitos dos fármacos , Gadolínio/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas da Mielina/deficiência , Proteínas da Mielina/genética , Células de Schwann/citologia , Células de Schwann/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Canais de Cátion TRPC/metabolismoRESUMO
Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot-Marie-Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Mutação de Sentido Incorreto , Proteínas da Mielina/química , Proteínas da Mielina/genética , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Doença de Charcot-Marie-Tooth/metabolismo , Cães , Humanos , Células Madin Darby de Rim Canino , Metais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas da Mielina/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Conformação Proteica , Estabilidade Proteica , Transporte Proteico , TermodinâmicaRESUMO
In the developing peripheral nervous system, axon-derived signals stimulate Schwann cells to undergo a global genetic reprogramming involving the cessation of cellular division and the upregulation of myelin genes. How such a comprehensive change in gene transcription is regulated is poorly understood. Here we report that BRG1/SMARCA4, the central helicase of the mammalian SWI/SNF-related chromatin remodeling complex, is required for Schwann cells to differentiate and form myelin, both in vitro and in vivo, in the mouse. BRG1 was highly activated in Schwann cells at early stages of myelination, and loss of the enzyme inhibited their differentiation and completely prevented myelin formation. Furthermore, we identify NF-κB as a key transcription factor that associates with the BRG1 complex in response to neuregulin 1 type III. During myelination, BRG1 was activated through the formation of a complex with NF-κB, and both proteins bound to the promoter region of Sox10, an inducer of myelination. These findings delineate a novel mechanism whereby axonal signals promote myelination through the remodeling of chromatin structure.
Assuntos
Diferenciação Celular/fisiologia , Cromatina/metabolismo , DNA Helicases/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Células de Schwann/fisiologia , Fatores de Transcrição/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Cromatina/fisiologia , Técnicas de Cocultura , DNA Helicases/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/fisiologia , Proteínas Nucleares/fisiologia , Ratos , Células de Schwann/citologia , Fatores de Transcrição/fisiologiaRESUMO
Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22.
Assuntos
Proteínas da Mielina/química , Dicroísmo Circular , Reagentes de Ligações Cruzadas , Glicerol , Humanos , Cinética , Micelas , Modelos Moleculares , Proteínas da Mielina/metabolismo , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Quaternária de Proteína , Termodinâmica , Resposta a Proteínas não DobradasRESUMO
Maspin, a 42-kDa non-classical serine protease inhibitor (serpin), is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation MS was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310 and Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin.
Assuntos
Epitélio Corneano/química , Processamento de Proteína Pós-Traducional , Serpinas/análise , Western Blotting , Linhagem Celular , Epitélio Corneano/metabolismo , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Humanos , Imunoprecipitação , Focalização Isoelétrica , Fosforilação , Serpinas/genética , Serpinas/metabolismo , Cicatrização/fisiologiaRESUMO
Maspin, an inhibitor of cell migration and a stimulator of adhesion of cells to the ECM, is synthesized and released by corneal keratocytes into the extracellular matrix. When the cornea is wounded, the quiescent stromal keratocytes underlying the wound undergo apoptosis and cells adjacent to this apoptotic area convert to fibroblasts or myofibroblasts. This study explores the effect of extracellular maspin on the plasminogen-plasminogen activator system of corneal stromal cells following wounding. Treatment of corneal fibroblasts and myofibroblasts with r-maspin increased extracellular but not cell-associated tissue-type plasminogen activator (tPA), urinary-type plasminogen activator (uPA) or plasminogen activator inhibitor-1 (PAI-1). Despite the extracellular increase in PAI-1, the net effect of maspin treatment was an increase in plasminogen activation. At physiological levels, maspin did not alter uPA or tPA mRNA levels, in these cells. The increase in pro and active uPA was due to decreased clearance in the presence of maspin for myofibroblasts but not for fibroblasts. The clearance of pro and active tPA was normal in fibroblasts indicating different mechanisms for the increase of these homologous enzymes in the two cell types. Increased generation of plasmin by maspin treated corneal stromal fibroblasts and myofibroblasts led to conversion of plasminogen to active plasmin degradation products and angiostatin-like molecules. This study suggests that extracellular maspin increased pro and active uPA and tPA released by corneal fibroblasts and myofibroblasts on the short time scale of 1-4 h, but by 24 h there was no increase over the levels produced without maspin. This augmentation of plasminogen activator activity increases plasmin activation and angiostatin generation. It further indicates that the effect of maspin on uPA and tPA levels is cell type dependent.
Assuntos
Substância Própria/citologia , Fibroblastos/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Ativadores de Plasminogênio/metabolismo , Inibidores de Serina Proteinase/farmacologia , Serpinas/farmacologia , Angiostatinas/metabolismo , Western Blotting , Células Cultivadas , Cicloeximida/farmacologia , Fibroblastos/metabolismo , Humanos , Miofibroblastos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/farmacologia , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , CicatrizaçãoRESUMO
Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032.
RESUMO
AIM: Amyotrophic lateral sclerosis (ALS) is a debilitating fatal neurodegenerative disorder. 90-95% of ALS cases are sporadic with no clear risk factors associated with the disease. Identification of biomarkers associated with ALS may lead to early detection and make it more amenable to therapeutic intervention. MATERIALS & METHODS: SILAC was used to quantitatively analyze the proteomes of ALS and control human fibroblasts. RESULTS: Out of total of 861 proteins identified, 33 were found to be differentially regulated. ApoB48 and Hsp20 were downregulated, while Fibulin-1 was upregulated. CONCLUSION: We report the differential regulation of these proteins in ALS fibroblasts, and their potential as novel biomarkers and possible drug targets for ALS.
Assuntos
Esclerose Lateral Amiotrófica/diagnóstico , Biomarcadores/metabolismo , Receptores de Lipoproteínas/metabolismo , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP20/metabolismo , Humanos , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Proteômica , Espectrometria de Massas em Tandem , Regulação para CimaRESUMO
PURPOSE: There is increasing evidence that cancer stem cells contribute to tumor progression and chemoresistance in a variety of malignancies, including brain tumors, leukemias, and breast carcinomas. In this study, we tested the hypothesis that retinoblastomas contain subpopulations of cells that exhibit cancer stem cell properties. METHODS: The following sources of retinoblastoma cells and tissues were examined for the presence of stem cell markers by immunocytochemistry: retinoblastoma tumors from mice transgenic for the SV40 T-antigen (driven by the beta-luteinizing hormone promoter), cell pellets of human Y79 and WERI-Rb27 retinoblastoma cell lines, and archival human retinoblastoma pathological specimens. Hoechst dye exclusion, mediated by the stem cell surface marker ABCG2 (ATP-binding cassette transporter, G2 subfamily), was assessed by flow cytometry in mouse tumors and WERI-Rb27 cells. RESULTS: Small numbers of retinoblastoma cells (less than 1%) exhibited immunoreactivity to stem cell markers ABCG2, aldehyde dehydrogenase 1 (ALDH1), MCM2 (minichromosome maintenance marker 2), SCA-1 (Stem cell antigen-1), and p63. Hoechst dye uptake in mouse tumors and WERI-Rb27 cells was enhanced by addition of 50 microM Verapamil, consistent with activity of the calcium-sensitive ABCG2 protein. Flow cytometric analysis confirmed the presence of small subpopulations of cells excluding Hoechst dye in mouse retinoblastoma tumors (0.3%) and WERI-Rb27 cells (0.1%) in a verapamil-sensitive manner. ABCG2 and ALDH1 positive cells were Hoechst-dim, as seen by dual labeling in vitro. CONCLUSIONS: Mouse and human retinoblastoma tumor cells contain a small subpopulation of cells that exhibit a cancer stem cell-like phenotype. Especially significant is the expression of ABCG2 in mouse and human tumor cells, a calcium-sensitive cell surface protein that not only acts to exclude Hoechst dye, but also confers resistance to over 20 different chemotherapeutic agents. These findings point to a heterogeneity in retinoblastoma tumors that may have significant impact on future treatment strategies.
Assuntos
Células-Tronco Neoplásicas/patologia , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Aldeído Desidrogenase/metabolismo , Família Aldeído Desidrogenase 1 , Animais , Antígenos Ly/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Genes Supressores de Tumor , Humanos , Técnicas Imunoenzimáticas , Isoenzimas/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Componente 2 do Complexo de Manutenção de Minicromossomo , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Retinal Desidrogenase , Neoplasias da Retina/metabolismo , Retinoblastoma/metabolismo , Transativadores/metabolismo , Fatores de Transcrição , Células Tumorais Cultivadas , Proteínas Supressoras de TumorAssuntos
Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/fisiologia , Serpinas/química , Serpinas/fisiologia , Animais , Antineoplásicos/farmacologia , Córnea/química , Neovascularização da Córnea/prevenção & controle , Neoplasias Oculares/tratamento farmacológico , Humanos , Conformação Proteica , Estrutura Terciária de Proteína , Inibidores de Serina Proteinase/farmacologia , Serpinas/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Withaferin A (WA) is a major bioactive compound isolated from the medicinal plant Withania somnifera Dunal, also known as "Ashwagandha". A number of published reports suggest various uses for WA including its function as an anti-inflammatory and anti-angiogenic drug molecule. The effects of WA at the molecular level in a cellular environment are not well understood. Knowledge of the molecular mechanism of action of WA could enhance its therapeutic value and may reveal novel pathways it may modulate. MATERIALS AND METHODS: In order to identify and characterize proteins affected by treatment with WA, we used SILAC- based proteomics analysis on a mouse microglial cell line (N9), which replicates phenotypic characteristics of primary microglial cells. RESULTS: Using stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry (MS), a total of 2300 unique protein groups were identified from three biological replicates, with significant expression changes in 32 non-redundant proteins. The top biological functions associated with these differentially expressed proteins include cell death and survival, free radical scavenging, and carbohydrate metabolism. Specifically, several heat shock proteins (Hsps) were found to be upregulated, which suggests that the chaperonic machinery might be regulated by WA. Furthermore, our study revealed several novel protein molecules that were not previously reported to be affected by WA. Among them, annexin A1, a key anti-inflammatory molecule in microglial cells was found to be downregulated. Hsc70, Hsp90α and Hsp105 were found to be upregulated. We also found sequestosome1/p62 (p62) to be upregulated. We performed Ingenuity Pathway Analysis (IPA) and found a number of pathways that were affected by WA treatment. CONCLUSIONS: SILAC-based proteomics analysis of a microglial cell model revealed several novel proteins whose expression is regulated by WA and probable pathways regulated by WA.
Assuntos
Microglia/efeitos dos fármacos , Vitanolídeos/farmacologia , Animais , Anexina A1/metabolismo , Metabolismo dos Carboidratos , Morte Celular , Linhagem Celular , Sobrevivência Celular , Proteínas de Choque Térmico/metabolismo , Marcação por Isótopo/métodos , Espectrometria de Massas , Camundongos , Microglia/metabolismo , ProteômicaRESUMO
TAR DNA binding protein (TDP43) is a DNA- and RNA-binding protein that is implicated in several neurodegenerative disorders termed as "TDP43 proteinopathies" including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and fronto-temporal lobe dementia (FTLD). We have developed an InCell Western (ICW) technique for screening TDP targeting drugs in 96 well plates. We tested 281 compounds and identified a novel compound hexachlorophene (referred to as B10) that showed potent reduction in TDP43 levels. The effect of B10 on TDP protein level was validated in two different cellular models: endogenous TDP43 expressing N9 microglial cells and TDP43-over-expressing HEK293 and HeLa cells. We also analyzed effect of B10 on various pathological forms of TDP such as the C25 cleaved fragment that localizes to the cytosol, insoluble high molecular weight species, and ALS-linked mutants. Our data suggest that B10 effectively reduces all forms of TDP. Overall, our data suggest that B10 could serve as a potential drug molecule for the treatment of AD, ALS and other TDP43 proteinopathies.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Descoberta de Drogas/métodos , Hexaclorofeno/farmacologia , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Hexaclorofeno/química , Humanos , Camundongos , Terapia de Alvo Molecular , Proteinopatias TDP-43/tratamento farmacológicoRESUMO
Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multi-domain protein that has been found to be mutated in patients with familial and sporadic Parkinson's disease, Alzheimer's disease and Crohn's disease. While the functions of LRRK2 are still largely unclear and mutations in LRRK2 are associated with adverse gain-of-function activities such as increased kinase activity, increased levels of LRRK2 alone are associated with toxicity in neurons. Consequently, exploring mechanisms to decrease levels of LRRK2 using pharmacological inhibitors would be highly advantageous. Previous work has shown that the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Cdc37 interact with and stabilize LRRK2. In the current study, we explore the regulation of LRRK2 by withaferin A (WA), a potent inhibitor of the interaction between Hsp90 and Cdc37. We report that treatment of the microglial cell line N9 with WA causes a decrease in cellular levels of LRRK2 in a dose- and time-dependent manner. We also find that treatment with WA disrupts the interaction between Hsp90, its co-chaperone Cdc37 and LRRK2, which leads to the destabilization and decreased levels of LRRK2. Additionally, treatment with celastrol, which is also an inhibitor of the Hsp90-Cdc37 complex, decreased LRRK2 levels. Interestingly, treatment with WA in the presence of celastrol enhanced the clearance of LRRK2. Overall, our data suggest that LRRK2 levels can be regulated by targeting the Hsp90-Cdc37 complex, which may have implications in the search for therapeutic strategies for Alzheimer's disease, Parkinson's disease and other LRRK2 proteinopathies.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Vitanolídeos/farmacologia , Animais , Linhagem Celular , Camundongos , Ligação Proteica/efeitos dos fármacosRESUMO
We previously discovered that one specific scalemic preparation of myricanol (1), a constituent of Myrica cerifera (bayberry/southern wax myrtle) root bark, could lower the levels of the microtubule-associated protein tau (MAPT). The significance is that tau accumulates in a number of neurodegenerative diseases, the most common being Alzheimer's disease (AD). Herein, a new synthetic route to prepare myricanol using a suitable boronic acid pinacol ester intermediate is reported. An X-ray crystal structure of the isolated myricanol (1) was obtained and showed a co-crystal consisting of (+)-aR,11S-myricanol (2) and (-)-aS,11R-myricanol (3) coformers. Surprisingly, 3, obtained from chiral separation from 1, reduced tau levels in both cultured cells and ex vivo brain slices from a mouse model of tauopathy at reasonable mid-to-low micromolar potency, whereas 2 did not. SILAC proteomics and cell assays revealed that 3 promoted tau degradation through an autophagic mechanism, which was in contrast to that of other tau-lowering compounds previously identified by our group. During the course of structure-activity relationship (SAR) development, we prepared compound 13 by acid-catalyzed dehydration of 1. 13 had undergone an unexpected structural rearrangement through the isomyricanol substitution pattern (e.g., 16), as verified by X-ray structural analysis. Compound 13 displayed robust tau-lowering activity, and, importantly, its enantiomers reduced tau levels similarly. Therefore, the semisynthetic analogue 13 provides a foundation for further development as a tau-lowering agent without its SAR being based on chirality.
Assuntos
Diarileptanoides/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas tau/metabolismo , Animais , Autofagia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Técnicas de Química Sintética , Diarileptanoides/isolamento & purificação , Diarileptanoides/farmacologia , Epitopos/metabolismo , Células HEK293/efeitos dos fármacos , Humanos , Masculino , Camundongos Transgênicos , Estrutura Molecular , Terapia de Alvo Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Estereoisomerismo , Tauopatias/tratamento farmacológico , Tauopatias/genética , Tauopatias/metabolismo , Proteínas tau/genética , Proteínas tau/imunologiaRESUMO
Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down-regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation is involved in the mechanism of down-regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-beta1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for Western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum-free defined medium, FGF-2 and TGF-beta1 were hypermethylated. Down-regulation of maspin synthesis was also associated with histone H3 dimethylation at lysine 9. Both maspin mRNA and protein were re-expressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down-regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.