Application of proteomics to graft-versus-host disease: from biomarker discovery to potential clinical applications.

INTRODUCTION: Graft-versus-host disease (GVHD) is a frequent and potentially life-threatening complication that occurs in many patients who undergo hematopoietic stem cell transplantation. In an effort to develop blood and tissue-based biochemical assays for GVHD diagnosis, high throughput proteomic platforms have been widely utilized for the identification and validation of disease biomarkers for both acute and chronic GVHD. Areas covered: This article reviews biomarker research findings on acute and chronic GVHD ascertained by studying peripheral blood, urine and saliva that gives biological information on systemic or localized disease. While the primary focus of GVHD biomarker discovery has been on identification and validation of prognostic and predictive biomarkers that might allow stratification of disease risk, molecular biomarkers that might aid patient diagnosis and/or response to treatment have also been reported. Expert commentary: Unbiased as well as targeted proteomic studies of acute and chronic GVHD have identified some distinguishing features of the two diseases especially the role of certain immune cell populations. A combination of patient risk stratification using panels of biomarkers and the application of novel targeted therapeutics should help to reduce the burden of GVHD, and hence improve the quality of life for patients following hematopoietic stem cell transplantation.

Unstimulated Saliva-Related Caries Risk Factors in Individuals with Cystic Fibrosis: A Cross-Sectional Analysis of Unstimulated Salivary Flow, pH, and Buffering Capacity.

Salivary flow rate, pH, and buffering capacity are associated with dental caries, but studies from the cystic fibrosis (CF) literature are inconclusive regarding these salivary factors and caries. The aim of this study was to evaluate these factors and their associations with dental caries in individuals with CF. Unstimulated whole saliva was collected from individuals aged 6-20 years at Seattle Children's Hospital CF Clinic, USA (n = 83). Salivary flow rate was measured in milliliters per minute. Salivary pH was assessed using a laboratory pH meter. Buffering capacity was assessed by titration with HCl. The outcome measure was caries prevalence, defined as the number of decayed, missing, or filled primary and permanent tooth surfaces. Spearman's rank correlation coefficient and the t test were used to test for bivariate associations. Multiple variable linear regression models were used to (1) run confounder-adjusted analyses and (2) assess for potential interactions. There was no significant association between salivary flow rate or buffering capacity and caries prevalence. There was a significant negative association between salivary pH and caries prevalence, but this association was no longer significant after adjusting for age. There was no significant interaction between salivary flow rate and buffering capacity or between antibiotic use and the 3 salivary factors. Our results indicate that unstimulated salivary factors are not associated with dental caries prevalence in individuals with CF. Future studies should investigate other potential saliva-related caries risk factors in individuals with CF such as cariogenic bacteria levels, salivary host defense peptide levels, and medication use.

Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.

Ichthyosis vulgaris (OMIM 146700) is the most common inherited disorder of keratinization and one of the most frequent single-gene disorders in humans. The most widely cited incidence figure is 1 in 250 based on a survey of 6,051 healthy English schoolchildren. We have identified homozygous or compound heterozygous mutations R501X and 2282del4 in the gene encoding filaggrin (FLG) as the cause of moderate or severe ichthyosis vulgaris in 15 kindreds. In addition, these mutations are semidominant; heterozygotes show a very mild phenotype with incomplete penetrance. The mutations show a combined allele frequency of approximately 4% in populations of European ancestry, explaining the high incidence of ichthyosis vulgaris. Profilaggrin is the major protein of keratohyalin granules in the epidermis. During terminal differentiation, it is cleaved into multiple filaggrin peptides that aggregate keratin filaments. The resultant matrix is cross-linked to form a major component of the cornified cell envelope. We find that loss or reduction of this major structural protein leads to varying degrees of impaired keratinization.

Proteomic analysis of saliva from patients with oral chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is an immune-mediated disorder and is the major long-term complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The oral mucosa, including the salivary glands, is affected in the majority of patients with cGVHD; however, at present there is only a limited understanding of disease pathobiology. In this study, we performed a quantitative proteomic analysis of saliva pooled from patients with and without oral cGVHD-cGVHD(+) and cGVHD(-), respectively-using isobaric tags for relative and absolute quantification labeling, followed by tandem mass spectrometry. Among 249 salivary proteins identified by tandem mass spectrometry, 82 exhibited altered expression in the oral cGVHD(+) group compared with the cGVHD(-) group. Many of the identified proteins function in innate or acquired immunity, or are associated with tissue maintenance functions, such as proteolysis or the cytoskeleton. Using ELISA immunoassays, we further confirmed that 2 of these proteins, IL-1 receptor antagonist and cystatin B, showed decreased expression in patients with active oral cGVHD (P < .003). Receiver operating curve characteristic analysis revealed that these 2 markers were able to distinguish oral cGVHD with a sensitivity of 85% and specificity of 60%, and showed slightly better discrimination in newly diagnosed patients evaluated within 12 months of allo-HSCT (sensitivity, 92%; specificity 73%). In addition to identifying novel potential salivary cGVHD biomarkers, our study demonstrates that there is coordinated regulation of protein families involved in inflammation, antimicrobial defense, and tissue protection in oral cGVHD that also may reflect changes in salivary gland function and damage to the oral mucosa.

Caspase-14 protects against epidermal UVB photodamage and water loss.

Caspase-14 belongs to a conserved family of aspartate-specific proteinases. Its expression is restricted almost exclusively to the suprabasal layers of the epidermis and the hair follicles. Moreover, the proteolytic activation of caspase-14 is associated with stratum corneum formation, implicating caspase-14 in terminal keratinocyte differentiation and cornification. Here, we show that the skin of caspase-14-deficient mice was shiny and lichenified, indicating an altered stratum-corneum composition. Caspase-14-deficient epidermis contained significantly more alveolar keratohyalin F-granules, the profilaggrin stores. Accordingly, caspase-14-deficient epidermis is characterized by an altered profilaggrin processing pattern and we show that recombinant caspase-14 can directly cleave profilaggrin in vitro. Caspase-14-deficient epidermis is characterized by reduced skin-hydration levels and increased water loss. In view of the important role of filaggrin in the structure and moisturization of the skin, the knockout phenotype could be explained by an aberrant processing of filaggrin. Importantly, the skin of caspase-14-deficient mice was highly sensitive to the formation of cyclobutane pyrimidine dimers after UVB irradiation, leading to increased levels of UVB-induced apoptosis. Removal of the stratum corneum indicate that caspase-14 controls the UVB scavenging capacity of the stratum corneum.

Reduced expression of epidermal growth factor receptor, E-cadherin, and occludin in the skin of flaky tail mice is due to filaggrin and loricrin deficiencies.

Disruption of skin barrier function leads to increases in the percutaneous transfer of allergens and the incidence of atopic dermatitis. Flaky tail (Flg(ft)) mice have been used as a model of atopic dermatitis with skin barrier dysfunction. Although Flg(ft) mice are known to have filaggrin mutation, the mechanism responsible for the skin barrier dysfunction that they display needs to be determined, especially for the roles of epidermal adhesion and junction proteins. Herein, we report the decreased expression of epidermal growth factor receptor (EGFR), E-cadherin, occludin, and SIRT1 in the skin of Flg(ft) mice, compared with those in C57BL/6J mice. Administration of N-acetyl-L-cysteine, an antioxidant, in the drinking water improved these protein expressions in the skin of Flg(ft) mice. Notably, we discovered that loricrin expression was suppressed in Flg(ft) mice. In vitro experiments showed that filaggrin small interfering RNA, loricrin small interfering RNA, or SIRT1 inhibitor sirtinol suppressed the expression levels of EGFR, E-cadherin, and occludin in a human immortalized keratinocyte cell line (HaCaT cells). Our findings suggest that the observed reductions in EGFR, E-cadherin, and occludin expression were due to filaggrin deficiency accompanied with subsequent loricrin deficiency and disruption of the SIRT1 pathway in the skin of Flg(ft) mice.

Filaggrin genotype in ichthyosis vulgaris predicts abnormalities in epidermal structure and function.

Although it is widely accepted that filaggrin (FLG) deficiency contributes to an abnormal barrier function in ichthyosis vulgaris and atopic dermatitis, the pathomechanism of how FLG deficiency provokes a barrier abnormality in humans is unknown. We report here that the presence of FLG mutations in Caucasians predicts dose-dependent alterations in epidermal permeability barrier function. Although FLG is an intracellular protein, the barrier abnormality occurred solely via a paracellular route in affected stratum corneum. Abnormal barrier function correlated with alterations in keratin filament organization (perinuclear retraction), impaired loading of lamellar body contents, followed by nonuniform extracellular distribution of secreted organelle contents, and abnormalities in lamellar bilayer architecture. In addition, we observed reductions in corneodesmosome density and tight junction protein expression. Thus, FLG deficiency provokes alterations in keratinocyte architecture that influence epidermal functions localizing to the extracellular matrix. These results clarify how FLG mutations impair epidermal permeability barrier function.

Association of a genetic polymorphism (-44 C/G SNP) in the human DEFB1 gene with expression and inducibility of multiple beta-defensins in gingival keratinocytes.

BACKGROUND: Human beta-defensins (hBDs) are antimicrobial peptides with a role in innate immune defense. Our laboratory previously showed that a single nucleotide polymorphism (SNP) in the 5' untranslated region of the hBD1 gene (DEFB1), denoted -44 (rs1800972), is correlated with protection from oral Candida. Because this SNP alters the putative mRNA structure, we hypothesized that it alters hBD1 expression. METHODS: Transfection of reporter constructs and evaluation of antimicrobial activity and mRNA expression levels in keratinocytes from multiple donors were used to evaluate the effect of this SNP on constitutive and induced levels of expression. RESULTS: Transfection of CAT reporter constructs containing the 5' untranslated region showed that the -44 G allele yielded a 2-fold increase in CAT protein compared to other common haplotypes suggesting a cis effect on transcription or translation. The constitutive hBD1 mRNA level in human oral keratinocytes was significantly greater in cells from donors with the -44 GG genotype compared to those with the common CC genotype. Surprisingly, the hBD3 mRNA level as well as antimicrobial activity of keratinocyte extracts also correlated with the -44 G allele. Induced levels of hBD1, hBD2, and hBD3 mRNA were evaluated in keratinocytes challenged with Toll-like receptor 2 and 4 ligands, interleukin-1beta, TNFalpha, and interferon-gamma (IFNgamma). In contrast to constitutive expression levels, IFNgamma-induced keratinocyte hBD1 and hBD3 mRNA expression was significantly greater in cells with the common CC genotype, but there was no clear correlation of genotype with hBD2 expression. CONCLUSION: The DEFB1 -44 G allele is associated with an increase in overall constitutive antimicrobial activity and expression of hBD1 and hBD3 in a manner that is consistent with protection from candidiasis, while the more common C allele is associated with IFNgamma inducibility of these beta-defensins and is likely to be more protective in conditions that enhance IFNgamma expression such as chronic periodontitis. These results suggest a complex relationship between genetics and defensin expression that may influence periodontal health and innate immune responses.

Age-related heterogeneity in dental caries and associated risk factors in individuals with cystic fibrosis ages 6-20 years: A pilot study.

BACKGROUND: The literature conflicts regarding dental caries risk in cystic fibrosis (CF) relative to controls. METHODS: Prospective, observational study of age-related heterogeneity in caries rates and potential risk factors in individuals with CF ages 6-20 at a single clinic in Washington state (N=85). Caries rates for enrolled CF participants and historical controls from NHANES were compared using cubic spline regression models. Generalized linear regression models identified correlates of age and caries in CF. RESULTS: Children ages 6-9 with CF had significantly lower caries than controls (Holm's P<0.05). There was no difference for ages 10-20 by CF status (Holm's P>0.05). Various biological/intraoral, medical, and behavioral factors were associated with caries and age in CF. CONCLUSIONS: Younger children with CF may be protected from caries, but there is apparent loss of protection in early adolescence associated with multiple risk factors. Additional studies are needed to confirm these findings.

Biology of chronic graft-vs-host disease: Immune mechanisms and progress in biomarker discovery.

Chronic graft-vs-host disease (cGVHD) is the leading cause of long-term morbidity and mortality following allogeneic hematopoietic stem cell transplantation. It presents as a chronic inflammatory and sclerotic autoimmune-like condition that most frequently affects the skin, oral mucosa, liver, eyes and gastrointestinal tract. Both clinical and animal studies have shown that multiple T cell subsets including Th1, Th2, Th17, T follicular helper cells and regulatory T-cells play some role in cGVHD development and progression; B cells also play an important role in the disease including the production of antibodies to HY and nuclear antigens that can cause serious tissue damage. An array of cytokines and chemokines produced by different types of immune cells also mediate tissue inflammation and damage of cGVHD target tissues such as the skin and oral cavity. Many of these same immune regulators have been studied as candidate cGVHD biomarkers. Recent studies suggest that some of these biomarkers may be useful for determining disease prognosis and planning long-term clinical follow-up of cGVHD patients.

TRPC channel expression during calcium-induced differentiation of human gingival keratinocytes.

BACKGROUND: Extracellular calcium is an important regulator of keratinocyte differentiation. An increase in intracellular calcium ion concentration is required for activation of calcium-induced keratinocyte differentiation. The signaling elements in this differentiation response include the calcium sensing receptor, phospholipase C, release of calcium ions from intracellular stores, and store-operated calcium channels. Nothing is currently known about the calcium-entry channels activated by the increase in external calcium. However, canonical transient receptor potential (TRPC) channels have been identified as store-operated calcium channels in several tissues. OBJECTIVE: To examine the expression of TRPC channels in human gingival keratinocytes (HGKs) in primary culture under both low calcium (basal) and high calcium (differentiating) conditions, and in gingival tissue. METHODS: TRPC channel expression was evaluated via RT-PCR, Western blots, and immunohistology. RESULTS: TRPC1, TRPC5, TRPC6 and TRPC7 mRNAs were detected in undifferentiated keratinocytes. Their levels initially increased, then decreased during calcium-induced differentiation. TRPC1 and TRPC6 protein expression reflected these changes. CONCLUSION: TRPC channels are present in both proliferating and differentiating keratinocytes in primary culture and in gingival tissue. The above expression patterns suggest that these channels may be involved in calcium-induced differentiation of keratinocytes.

A search for CDKN2A/p16INK4a mutations in melanocytic nevi from patients with melanoma and spouse controls by use of laser-captured microdissection.

OBJECTIVE: To determine the frequency at which the CDKN2A coding region is mutated in the atypical nevi of persons with sporadic melanoma. DESIGN: DNA samples, isolated by laser-captured microdissection of atypical nevi from 10 patients with newly incident cases of sporadic melanoma and their spouses as matched controls, were used as templates for nested polymerase chain reaction amplification of CDKN2A exons 1 and 2. RESULTS: No point mutations in the coding region of CDKN2A were observed in any of the melanocytic nevi. CONCLUSIONS: Point mutations in CDKN2A are an uncommon event in the atypical nevi of persons with melanoma. As such, the data may support a hypothesis of melanocytic nevus histogenesis, in which the melanocytic nevus and malignant melanoma represent separate, pleiotropic pathways resulting from common stimuli, such as genomic damage from UV radiation.

Tetracycline-regulated gene expression in epidermal keratinocytes.

The tetracycline-regulated expression system developed by Gossen and Bujard is a powerful genetic tool that permits the expression of any gene construct introduced into either cultured cells or transgenic animals to be precisely controlled. It involves two components, a regulatory component based on the prokaryotic tetracycline repressor (TetR) and a response plasmid that expresses the gene of interest under control of the tetracycline-response element. In this paper, we review the Tet system methodology, discuss the available vector systems, and describe how to prepare and characterize keratinocyte cell lines that express a gene under tetracycline control. The methodology involves the development of stable cell lines expressing the TetR protein (either tTA or rtTA, expressed as a fusion with the VP16 activation domain), and a second set of double-stable cell lines that contain both TetR and the response plasmid (tetracycline-response element-gene X) expressed under tetracycline control. As an example of this methodology, we discuss our recently developed keratinocyte cell lines that express human filaggrin in a tetracycline- regulated manner. This technique, now also available in retrovirus and adenovirus-based vectors, is applicable both to the study of genes that are toxic to cells and more generally to understand how genes regulate cell structure/function, growth, and differentiation.

Crystal Structure of Human Profilaggrin S100 Domain and Identification of Target Proteins Annexin II, Stratifin, and HSP27.

The fused-type S100 protein profilaggrin and its proteolytic products including filaggrin are important in the formation of a normal epidermal barrier; however, the specific function of the S100 calcium-binding domain in profilaggrin biology is poorly understood. To explore its molecular function, we determined a 2.2 Å-resolution crystal structure of the N-terminal fused-type S100 domain of human profilaggrin with bound calcium ions. The profilaggrin S100 domain formed a stable dimer, which contained two hydrophobic pockets that provide a molecular interface for protein interactions. Biochemical and molecular approaches demonstrated that three proteins, annexin II/p36, stratifin/14-3-3 sigma, and heat shock protein 27, bind to the N-terminal domain of human profilaggrin; one protein (stratifin) co-localized with profilaggrin in the differentiating granular cell layer of human skin. Together, these findings suggest a model where the profilaggrin N-terminus uses calcium-dependent and calcium-independent protein-protein interactions to regulate its involvement in keratinocyte terminal differentiation and incorporation into the cornified cell envelope.

Functional analysis of the profilaggrin N-terminal peptide: identification of domains that regulate nuclear and cytoplasmic distribution.

Profilaggrin is expressed in the differentiating granular layer of epidermis and other stratified epithelia, where it forms a major component of cytoplasmic keratohyalin granules. It consists of two distinct domains, an N-terminal S100-like Ca2+- binding domain containing two EF-hands and multiple filaggrin units that aggregate keratin filaments in the stratum corneum. Here, we report structure-function studies of the N-terminal peptide from mouse, human, and rat profilaggrin. The profilaggrin N- terminal peptides of all species contain two S100-like EF-hands, bipartite nuclear localization sequences, and proprotein convertase cleavage sites. The nuclear localization signals in human and mouse profilaggrin were shown to be functional by transfection of epithelial cells and depended on the absence of filaggrin sequences. The nuclear localization of the processed (free) N-terminal peptide of human profilaggrin is consistent with immunolocalization findings in normal human skin and in parakeratotic skin disorders, which exhibit nuclear staining of granular and/or cornified layers. The mouse profilaggrin N-terminus undergoes proteolytic processing in two steps, first releasing an N-terminal peptide containing some filaggrin sequence and finally the free N-terminus of 28-30 kDa; these peptides have cytoplasmic and nuclear distributions, respectively, when expressed in transfected cells. The N-terminal processing may occur prior to or simultaneously with the proteolytic processing of the polyfilaggrin domain. The nuclear accumulation of the profilaggrin N-terminal peptide in epidermis and in transfected cells strongly suggests a calcium-dependent nuclear function for the profilaggrin N-terminus during epidermal terminal differentia tion when the free N-terminus is released from profilaggrin by specific proteolysis.

Analysis of epidermal-type transglutaminase (transglutaminase 3) in human stratified epithelia and cultured keratinocytes using monoclonal antibodies.

BACKGROUND: Epidermal-type transglutaminase (TGase 3) is involved in the cross-linking of structural proteins in the epidermis, which results in the formation of the cornified envelope. TGase 3 is activated by limited proteolysis of a 77 kDa zymogen during keratinocyte differentiation. OBJECTIVE: To characterize the expression of TGase 3 in human epidermis and cultured keratinocytes, we established specific monoclonal antibodies against the TGase 3. METHODS: Recombinant proteins for human TGase 3 produced in bacteria and baculovirus-infected insect cells were purified as an antigen. Hybridomas are established and used for characterization of expression in epidermis and keratinocytes. RESULTS: Four antibodies were generated against recombinant human TGase 3, which reacted with the 77 kDa zymogen and in some cases either the 47 or 30 kDa active proteolytic fragments. In human epidermis and cultured keratinocytes, only the zymogen form of TGase 3 was detected. Immunohistochemical analysis of the skin revealed that the enzyme is present in the cells of the granular and cornified layers consistent with its role in cornified envelope formation. In cultured keratinocytes, TGase 3 was expressed in differentiating cells coincident with profilaggrin and keratin 10 expressions. CONCLUSION: Using monoclonal antibody against human TGase 3, we showed the expression of TGase 3 in upper layers of epidermis. TGase 3 displayed a diffuse cytoplasmic distribution in vitro consistent with its proposed role in the early phase of cornified cell envelope assembly in the cytoplasm.

Making sense of the epithelial barrier: what molecular biology and genetics tell us about the functions of oral mucosal and epidermal tissues.

The epidermis of skin and the oral mucosa are highly specialized stratified epithelia that function to protect the body from physical and chemical damage, infection, dehydration, and heat loss. To maintain this critical barrier, epithelial tissues undergo constant renewal and repair. Epithelial cells (keratinocytes) undergo a program of terminal differentiation, expressing a set of structural proteins, keratins, which assemble into filaments and function to maintain cell and tissue integrity. Two types of cell adhesion structures, desmosomes and hemidesmosomes, function to glue keratinocytes to one another and to the basement membrane, and connect the keratin cytoskeleton to the cell surface. Keratinizing epithelia such as the epidermis and oral gingiva that have to withstand severe physical and chemical forces produce a toughened structure, the cornified cell envelope. This envelope is a major component of the epithelial barrier at the tissue surface. This article summarizes our current understanding of the structure and function of these different cellular components and discusses various genetic and acquired diseases that alter tissue integrity and barrier function. We also highlight recent work demonstrating how loss or attenuation of certain proteases can lead to early onset periodontitis and tooth loss as well as other epithelial abnormalities.

Interaction of the profilaggrin N-terminal domain with loricrin in human cultured keratinocytes and epidermis.

The relationship between the two coexpressed differentiation markers, profilaggrin and loricrin, is not clear right now. In this study, we explored the interaction of profilaggrin N-terminal domain (PND) with loricrin in keratinocytes and epidermis. Confocal immunofluorescence microscopic analysis of human epidermis showed that PND colocalized with loricrin. Loricrin nucleofected into HaCaT cells colocalized with PND in the nucleus and cytoplasm. The PND localizes to both the nucleus and cytoplasm of epidermal granular layer cells. Nucleofected PND also colocalized with keratin 10 (K10) in the nucleus and cytoplasm. Immunoelectron microscopic analysis of human epidermis confirmed the findings in nucleofected keratinocytes. Yeast two-hybrid assays showed that the B domain of human and mouse PND interacted with loricrin. The glutathione S-transferase (GST) pull-down analysis using recombinant GST-PND revealed that PND interacted with loricrin and K10. Knockdown of PND in an organotypic skin culture model caused loss of filaggrin expression and a reduction in both the size and number of keratohyalin granules, as well as markedly reduced expression of loricrin. Considering that expression of PND is closely linked to keratinocyte terminal differentiation, we conclude that PND interacts with loricrin and K10 in vivo and that these interactions are likely to be relevant for cornified envelope assembly and subsequent epidermal barrier formation.

Caspase-14 is required for filaggrin degradation to natural moisturizing factors in the skin.

Caspase-14 is a protease that is mainly expressed in suprabasal epidermal layers and activated during keratinocyte cornification. Caspase-14-deficient mice display reduced epidermal barrier function and increased sensitivity to UVB radiation. In these mice, profilaggrin, a protein with a pivotal role in skin barrier function, is processed correctly to its functional filaggrin (FLG) repeat unit, but proteolytic FLG fragments accumulate in the epidermis. In wild-type stratum corneum, FLG is degraded into free amino acids, some of which contribute to generation of the natural moisturizing factors (NMFs) that maintain epidermal hydration. We found that caspase-14 cleaves the FLG repeat unit and identified two caspase-14 cleavage sites. These results indicate that accumulation of FLG fragments in caspase-14(-/-) mice is due to a defect in the terminal FLG degradation pathway. Consequently, we show that the defective FLG degradation in caspase-14-deficient skin results in substantial reduction in the amount of NMFs, such as urocanic acid and pyrrolidone carboxylic acid. Taken together, we identified caspase-14 as a crucial protease in FLG catabolism.