Retention of corneodesmosomes and increased expression of protease inhibitors in dandruff.

BACKGROUND: Dandruff is a common, relapsing and uncomfortable scalp condition affecting a large proportion of the global population. The appearance of flakes on the scalp and in the hair line, and associated itch are thought to be consequences of a damaged skin barrier, altered corneocyte cohesion and abnormal desquamation in dandruff. The balance between skin proteases and protease inhibitors is essential for driving the key events, including corneodesmosome degradation, in the desquamation process and to maintain stratum corneum (SC) barrier integrity. OBJECTIVES: To investigate the distribution of corneodesmosomes, the key component of the SC cohesivity and barrier function, and the protease inhibitors lympho-epithelial Kazal-type-related inhibitor (LEKTI-1) and squamous cell carcinoma antigen (SCCA1) in the scalp of dandruff-affected participants. METHODS: The methods utilized were immunohistochemistry, scanning immunoelectron microscopy, phase-contrast microscopy, Western blotting and serine protease activity assay on tape-stripped SC or scalp skin biopsies. RESULTS: In SC samples from healthy subjects, corneodesmosomes were peripherally located in the corneocytes. In samples of dandruff lesions, corneodesmosomes were located both peripherally and on the entire surface area of the corneocytes. LEKTI-1 and SCCA1 protein levels and parakeratosis were found to be highly elevated in the lesional samples. CONCLUSIONS: The persistence of nonperipheral corneodesmosomes is a characteristic feature of the perturbed desquamation seen in dandruff. The increased expression levels of LEKTI-1 and SCCA1 are consistent with the view that the dandruff condition is characterized by an imbalance in protease-protease inhibitor interaction in the SC.

Direct evidence that PKCα positively regulates wound re-epithelialization: correlation with changes in desmosomal adhesiveness.

Non-healing wounds cause considerable patient morbidity and represent a significant economic burden. Central to wound repair is re-epithelialization, a crucial process involving the modulation of cell adhesion to allow keratinocyte migration to cover the exposed underlying tissues. The cellular mechanisms regulating the earliest stages of re-epithelialization are unclear. We present the first direct evidence that protein kinase Cα (PKCα) plays an important role in regulating wound re-epithelialization. In PKCα(-/-) mice re-epithelialization is delayed, while in novel bitransgenic mice over-expressing constitutively active PKCα it is accelerated. These effects are not due to changes in keratinocyte proliferation, apoptosis or intrinsic cell motility. Instead, they correlate with changes in desmosomal adhesiveness, delay being preceded by retained desmosomal hyper-adhesiveness and acceleration with a rapid switch to desmosomal Ca(2+) -dependence. We demonstrate mechanistic conservation in acute human wounds where PKCα localizes to wound edge desmosomes, which become Ca(2+) -dependent. However, in chronic wounds PKCα remains cytoplasmic and desmosomes fail to switch from the hyper-adhesive state. These results throw new mechanistic light on the earliest stages of wound re-epithelialization and suggest activation of PKCα as a new therapeutic strategy for non-healing wounds.

Regulation of small GTPases at epithelial cell-cell junctions.

Small GTPases of the Rho family (RhoA, Rac1, and Cdc42) and the Ras family GTPase Rap1 are essential for the assembly and function of epithelial cell-cell junctions. Through their downstream effectors, small GTPases modulate junction formation and stability, primarily by orchestrating the polymerization and contractility of the actomyosin cytoskeleton. The major upstream regulators of small GTPases are guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Several GEFs and a few GAPs have been localized at epithelial junctions, and bind to specific junctional proteins. Thus, junctional proteins can regulate small GTPases at junctions, through their interactions with GEFs and GAPs. Here we review the current knowledge about the mechanisms of regulation of small GTPases by junctional proteins. Understanding these mechanisms will help to clarify at the molecular level how small GTPases control the morphogenesis and physiology of epithelial tissues, and how they are disregulated in disease.

Role of Rho GTPases in desmosomal adhesion and pemphigus pathogenesis.

Desmosomes are distinct intercellular contacts essential to the integrity of epithelial tissues and the heart muscle. This function is impaired in the disease pemphigus, in which patients develop autoantibodies against the cadherin-type desmosomal core proteins desmogleins. Autoantibody binding induces loss of cell-cell adhesion leading to blisters within the epidermis and mucous membranes. Despite the relevance of desmosomes for integrity of such essential organs as the skin, data on the regulation of desmosome assembly and maintenance and desmosome-mediated adhesion are only slowly emerging. Small guanosine triphosphatases (GTPases) of the Rho family have long been established as regulators of other cell junctions such as adherens junctions, but also have been implicated in participating in the formation of desmosomes. In this short review we summarize two papers from our group dealing with the role of Rho family GTPases for desmosomal adhesion and pemphigus and discuss these data integrating novel work recently published.

Involvement of corneodesmosome degradation and lamellar granule transportation in the desquamation process.

Desquamation in the mammalian skin is a well-balanced process of producing corneocytes and shedding them from the surface of the skin. The corneodesmosome, which is a modified desmosome, is the main adhesive structure in the cornified cell layer. The major extracellular constituents of corneodesmosomes are desmoglein 1, desmocollin 1, and corneodesmosin. Proteases involved in the degradation of corneodesmosomes and their inhibitors are secreted from lamellar granules in the granular cell layer. Genetic defects in corneodesmosin and protease inhibitors result in accelerated desquamation and severe barrier impairment. Abnormalities in transportation and secretion of lamellar granules underlie ichthyosis seen in certain human diseases.

Plakophilin 2: a critical scaffold for PKC alpha that regulates intercellular junction assembly.

Plakophilins (PKPs) are armadillo family members related to the classical cadherin-associated protein p120(ctn). PKPs localize to the cytoplasmic plaque of intercellular junctions and participate in linking the intermediate filament (IF)-binding protein desmoplakin (DP) to desmosomal cadherins. In response to cell-cell contact, PKP2 associates with DP in plaque precursors that form in the cytoplasm and translocate to nascent desmosomes. Here, we provide evidence that PKP2 governs DP assembly dynamics by scaffolding a DP-PKP2-protein kinase C alpha (PKC alpha) complex, which is disrupted by PKP2 knockdown. The behavior of a phosphorylation-deficient DP mutant that associates more tightly with IF is mimicked by PKP2 and PKC alpha knockdown and PKC pharmacological inhibition, all of which impair junction assembly. PKP2 knockdown is accompanied by increased phosphorylation of PKC substrates, raising the possibility that global alterations in PKC signaling may contribute to pathogenesis of congenital defects caused by PKP2 deficiency.

Inhibition of Rho A activity causes pemphigus skin blistering.

The autoimmune blistering skin diseases pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are mainly caused by autoantibodies against desmosomal cadherins. In this study, we provide evidence that PV-immunoglobulin G (IgG) and PF-IgG induce skin blistering by interference with Rho A signaling. In vitro, pemphigus IgG caused typical hallmarks of pemphigus pathogenesis such as epidermal blistering in human skin, cell dissociation, and loss of desmoglein 1 (Dsg 1)-mediated binding probed by laser tweezers. These changes were accompanied by interference with Rho A activation and reduction of Rho A activity. Pemphigus IgG-triggered keratinocyte dissociation and Rho A inactivation were p38 mitogen-activated protein kinase dependent. Specific activation of Rho A by cytotoxic necrotizing factor-y abolished all pemphigus-triggered effects, including keratin retraction and release of Dsg 3 from the cytoskeleton. These data demonstrate that Rho A is involved in the regulation of desmosomal adhesion, at least in part by maintaining the cytoskeletal anchorage of desmosomal proteins. This may open the possibility of pemphigus treatment with the epidermal application of Rho A agonists.

Corneodesmosomal cadherins are preferential targets of stratum corneum trypsin- and chymotrypsin-like hyperactivity in Netherton syndrome.

SPINK5 (serine protease inhibitor Kazal-type 5), encoding the protease inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor), is the defective gene in Netherton syndrome (NS), a severe inherited keratinizing disorder. We have recently demonstrated epidermal protease hyperactivity in Spink5(-/-) mice resulting in desmosomal protein degradation. Herein, we investigated the molecular mechanism underlying the epidermal defect in 15 patients with NS. We demonstrated that, in a majority of patients, desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) were dramatically reduced in the upper most living layers of the epidermis. These defects were associated with premature degradation of corneodesmosomes. Stratum corneum tryptic enzyme (SCTE)-like and stratum corneum chymotryptic enzyme (SCCE)-like activities were increased, suggesting that these proteases participate in the premature degradation of corneodesmosomal cadherins. SCTE and SCCE expression was extended to the cell layers where Dsg1 and Dsc1 immunostaining was reduced. In contrast, a subset of six patients with normal epidermal protease activity or residual LEKTI expression displayed apparently normal cadherin expression and less severe disease manifestations. This suggests a degree of correlation between cadherin degradation and clinical severity. This work further supports the implication of premature corneodesmosomal cadherin degradation in the pathogenesis of NS and provides evidence for additional factors playing a role in disease expression.

LEKTI is localized in lamellar granules, separated from KLK5 and KLK7, and is secreted in the extracellular spaces of the superficial stratum granulosum.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a putative serine protease inhibitor encoded by serine protease inhibitor Kazal-type 5 (SPINK5). It is strongly expressed in differentiated keratinocytes in normal skin but expression is markedly reduced or absent in Netherton syndrome (NS), a severe ichthyosis caused by SPINK5 mutations. At present, however, both the precise intracellular localization and biological roles of LEKTI are not known. To understand the functional role of LEKTI, we examined the localization of LEKTI together with kallikrein (KLK)7 and KLK5, possible targets of LEKTI, in the human epidermis, by confocal laser scanning microscopy and immunoelectron microscopy. In normal skin, LEKTI, KLK7, and KLK5 were all found in the lamellar granule (LG) system, but were separately localized. LEKTI was expressed earlier than KLK7 and KLK5. In NS skin, LEKTI was absent and an abnormal split in the superficial stratum granulosum was seen in three of four cases. Collectively, these results suggest that in normal skin the LG system transports and secretes LEKTI earlier than KLK7 and KLK5 preventing premature loss of stratum corneum integrity/cohesion. Our data provide new insights into the biological functions of LG and the pathogenesis of NS.

Degradation of corneodesmosome proteins by two serine proteases of the kallikrein family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7.

Corneodesmosin (CDSN), desmoglein 1 (DSG1), and desmocollin 1 (DSC1) are adhesive proteins of the extracellular part of the corneodesmosomes, the junctional structures that mediate corneocyte cohesion. The degradation of these proteins at the epidermis surface is necessary for desquamation. Two serine proteases of the kallikrein family synthesized as inactive precursors have been implicated in this process: the stratum corneum chymotryptic enzyme (SCCE/KLK7/hK7) and the stratum corneum tryptic enzyme (SCTE/KLK5/hK5). Here, we analyzed the capacity of these enzymes to cleave DSG1, DSC1, and epidermal or recombinant forms of CDSN, at an acidic pH close to that of the stratum corneum. SCCE directly cleaved CDSN and DSC1 but was unable to degrade DSG1. But incubation with SCTE induced degradation of the three corneodesmosomal components. Using the recombinant form of CDSN, either with its N-glycan chain or enzymatically deglycosylated, we also demonstrated that oligosaccharide residues do not protect CDSN against proteolysis by SCCE. Moreover, our results suggest that SCTE is able to activate the proform of SCCE. These results strongly suggest that the two kalikreins are involved in desquamation. A model is proposed for desquamation that could be regulated by a precisely controlled protease-protease inhibitor balance.

Molecular cloning and tissue expression of the murine analog to human stratum corneum chymotryptic enzyme.

Human stratum corneum chymotryptic enzyme (SCCE) may play a central part in epidermal homeostasis. Its proposed function is to catalyze the degradation of intercellular structures, including desmosomes, in the stratum corneum as part of the desquamation process. In order to facilitate physiologic and pathophysiologic studies on SCCE we have looked for the corresponding murine enzyme. A cDNA obtained by reverse transcription-polymerase chain reaction with total RNA prepared from mouse tails as starting material was cloned, and the expression of the corresponding mRNA studied. The murine cDNA showed 77% homology to human SCCE cDNA. It had an open-reading frame encoding a protein comprising 249 amino acids with 82% amino acid sequence homology to human SCCE including the conserved sequences of the catalytic traid of mammalian serine proteases. The murine protein was deduced to have a 21 amino acid signal peptide and a four amino acid propeptide ending with a tryptic cleavage site, followed by a sequence motif identical to the N-terminal amino acid sequence of native active human SCCE. As in human SCCE the P2 position of the propeptide was occupied by an acidic amino acid residue, and the position corresponding to the suggested bottom of the primary substrate specificity pouch occupied by an asparagine residue. Analyses of mouse tissues by reverse transcriptase-polymerase chain reaction showed high expression in the skin, low expression in lung, kidney, brain, heart, and spleen, and no expression in liver or skeletal muscle. In situ hybridization of mouse skin showed expression in high suprabasal keratinocytes and in the luminal parts of hair follicles. Our results strongly suggest that we have cloned the murine analog of human SCCE cDNA.

Cellular redistribution of protein tyrosine phosphatases LAR and PTPsigma by inducible proteolytic processing.

Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPsigma. PTPsigma biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPsigma underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCalpha. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPsigma cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and beta-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPsigma in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell-cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPsigma is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell-cell contacts.

Protein kinase C activation upregulates intercellular adhesion of alpha-catenin-negative human colon cancer cell variants via induction of desmosomes.

The alpha-catenin molecule links E-cadherin/ beta-catenin or E-cadherin/plakoglobin complexes to the actin cytoskeleton. We studied several invasive human colon carcinoma cell lines lacking alpha-catenin. They showed a solitary and rounded morphotype that correlated with increased invasiveness. These round cell variants acquired a more normal epithelial phenotype upon transfection with an alpha-catenin expression plasmid, but also upon treatment with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Video registrations showed that the cells started to establish elaborated intercellular junctions within 30 min after addition of TPA. Interestingly, this normalizing TPA effect was not associated with alpha-catenin induction. Classical and confocal immunofluorescence showed only minor TPA-induced changes in E-cadherin staining. In contrast, desmosomal and tight junctional proteins were dramatically rearranged, with a conversion from cytoplasmic clusters to obvious concentration at cell-cell contacts and exposition at the exterior cell surface. Electron microscopical observations revealed the TPA-induced appearance of typical desmosomal plaques. TPA-restored cell-cell adhesion was E-cadherin dependent as demonstrated by a blocking antibody in a cell aggregation assay. Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too. Remarkably, the combination of anti-E-cadherin and anti-desmoglein antibodies synergistically inhibited the TPA effect. Our studies show that it is possible to bypass the need for normal alpha-catenin expression to establish tight intercellular adhesion by epithelial cells. Apparently, the underlying mechanism comprises upregulation of desmosomes and tight junctions by activation of the PKC signaling pathway, whereas E-cadherin remains essential for basic cell-cell adhesion, even in the absence of alpha-catenin.

Evidence that stratum corneum chymotryptic enzyme is transported to the stratum corneum extracellular space via lamellar bodies.

Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered human serine proteinase that may be specific for keratinizing squamous epithelia. SCCE has properties compatible with a function in the degradation of intercellular cohesive structures during stratum corneum turnover and desquamation. SCCE is expressed in suprabasal keratinocytes. In this study, we demonstrate the subcellular localization of SCCE in the upper granular layer, in the stratum corneum of normal non-palmoplantar skin, and in cohesive parts of hypertrophic plantar stratum corneum, using immunoelectron microscopy of ultrathin cryosections labeled with SCCE-specific monoclonal antibodies detected with gold-labeled secondary antibodies. A narrow zone close to the transition between the granular and cornified layers showed positive SCCE staining after fixation. By means of immunoelectron microscopy, SCCE was found in association with structures resembling intracellular lamellar bodies in the uppermost granular cells and in similar structures undergoing extrusion to the extracellular space between the uppermost granular cells and the lowermost cornified cells. In the stratum corneum, the detected SCCE was confined to the extracellular space and was found in association with intact and partially degraded desmosomes, as well as in the parts of the extracellular space devoid of desmosomes. We conclude that SCCE may be stored in lamellar bodies in the stratum granulosum and transported via these structures to the stratum corneum extracellular space. The results further support the idea that the physiologic function of SCCE may be to catalyze the degradation of desmosomes in the stratum corneum during remodeling of the deeper layers of this tissue, and at a later stage serve as a prerequisite for desquamation.

Monoclonal antibody to desmosomal glycoprotein 1--a new epithelial marker for diagnostic pathology.

Desmosomes are intercellular adhesive junctions that occur in almost all epithelia and should therefore be useful as epithelial markers in tumour diagnosis. Here, we describe a monoclonal antibody, 32-2B, to a major desmosomal glycoprotein (dgl) which reacts with human tissues in paraffin sections. This antibody was tested for its ability to stain epithelia and tumours. It reacted with all epithelia tested and with every specimen of a wide range of carcinomas. It also stained meningiomas, another desmosome-containing tumour. It did not stain other types of tumours including lymphomas, melanomas, and various sarcomas, or normal tissues which lack desmosomes. These characteristics demonstrate that 32-2B is a reliable epithelial marker that may have a useful role in diagnostic histopathology.

Cytochemical localization of a "basic" ATPase to canine myocardial surface membrane.

Enzymatic properties of a canine cardiac muscle microsomal fraction were determined to localize in situ a "basic," divalent cation dependent adenosine triphosphatase (ATPase) by ultrastructural cytochemistry. The microsomal fraction had a buoyant density of 1.08--1.13 (20--30% [w/w] sucrose) and hydrolyzed adenosine triphosphate in the presence of Mg2+, Ca2+, Mn2+, or Co2+, but not in that of Sr2+ or Ni2+, under conditions that inhibited interfering (Na+ + K+)-ATPase and sarcoplasmic reticulum Ca2+-ATPase activities. "Basic" ATPase was localized in paraformaldehyde-fixed tissue in a medium containing Mg2+ or a high Ca2+ concentration (4 mM). A free Pb2+ concentration of less than 1 microM was used to capture enzymatically released phosphate anions. Electron-dense lead precipitates were present at the plasmalemma, T-system, and intercalated disc membranes with the exception of the nexus. These studies suggest that "basic" ATPase activity is associated with surface membrane structures of canine cardiac muscle.