Influence of Vitamin D on Corneal Epithelial Cell Desmosomes and Hemidesmosomes.

Purpose: We have observed noticably weak epithelial attachment in vitamin D receptor knockout mice (VDR KO) undergoing epithelial debridement. We hypothesized that VDR KO negatively affects corneal epithelial cell desmosomes and/or hemidesmosomes. Methods: Transcript levels of desmosome and hemidesmosome proteins in VDR KO corneas were assessed by qPCR. Western blotting and immunochemistry were used to detect proteins in cultured cells exposed to 1,25(OH)2D3 and 24R,25(OH)2D3. Results: VDR KO resulted in decreased corneal desmosomal desmoglein 1 (DSG1) and desmocollin 2 (DSC2) mRNA, and hemidesmosomal plectin mRNA. DSG1 and plectin protein expression were reduced in VDR KO corneas. DSG1 protein expression increased in VDR wild types (VDR WT) and VDR KO mouse primary epithelial cells (MPCEC) treated with 1,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 treatment resulted in increased plectin and integrin ß4 levels in VDR WT MPCEC, and decreased levels in VDR KO MPCEC. Treatment of human corneal epithelial cells (HCEC) with 1,25(OH)2D3 and 24R,25(OH)2D3 resulted in increased DSC2 and DSG1 protein expression. Plectin and integrin ß4 were only increased in 24R,25(OH)2D3 treated HCEC. Conclusions: VDR KO results in reduced desmosomal and hemidesmosomal mRNA and protein levels. 1,25(OH)2D3 and 24R,25(OH)2D3 increased DSG1 protein in all cells tested. For hemidesmosome proteins, 24R,25(OH)2D3 increased plectin and integrin ß4 protein expression in VDR WT and HCEC, with decreased expression in VDR KO MPCEC. Thus, vitamin D3 is involved in desmosome and hemidesmosome junction formation/regulation, and their decreased expression likely contributes to the loosely adherent corneal epithelium in VDR KO mice. Our data indicate the presence of a VDR-independent pathway.

Peptide coatings enhance keratinocyte attachment towards improving the peri-implant mucosal seal.

There is a critical need for preventing peri-implantitis as its prevalence has increased and dental implants lack features to prevent it. Research strategies to prevent peri-implantitis have focused on modifying dental implants to incorporate different antimicrobial agents. An alternative strategy consists of barring the expansion of the biofilm subgingivally by forming a long-lasting permucosal seal between the soft tissue and the implant surface. Here, we innovatively biofunctionalized titanium with bioinspired peptide coatings to strengthen biological interactions between epithelial cells and the titanium surface. We selected laminin 332- and ameloblastin-derived peptides (Lam, Ambn). Laminin 332 participates in the formation of hemidesmosomes by keratinocytes and promotes epithelial attachment around teeth; and ameloblastin, an enamel derived protein, is involved in tissue regeneration events following disruption of the periodontium. Lam, Ambn or combinations of both peptides were covalently immobilized on titanium discs. Successful immobilization of the peptides was confirmed by contact angle goniometry, X-ray photoelectron spectroscopy and fluorescent labelling of the peptides. Additionally, we confirmed the mechanical and thermochemical stability of the peptides on Ti substrates. Proliferation and hemidesmosome formation of human keratinocytes (TERT-2/OKF-6) were assessed by immunofluorescence labelling. The peptide-coated surfaces increased cell proliferation for up to 48 h in culture compared to control surfaces. Most importantly, formation of hemidesmosomes by keratinocytes was significantly increased on surfaces coated with Ambn + Lam peptides compared to control (p < 0.01) and monopeptide coatings (p < 0.005). Together, these results support the Ambn + Lam multipeptide coating as a promising candidate for inducing a permucosal seal around dental implants.

Vimentin-mediated regulation of cell motility through modulation of beta4 integrin protein levels in oral tumor derived cells.

Vimentin expression correlates well with migratory and invasive potential of the carcinoma cells. The molecular mechanism by which vimentin regulates cell motility is not yet clear. Here, we addressed this issue by depleting vimentin in oral squamous cell carcinoma derived cell line. Vimentin knockdown cells showed enhanced adhesion and spreading to laminin-5. However, we found that they were less invasive as compared to the vector control cells. In addition, signaling associated with adhesion behavior of the cell was increased in vimentin knockdown clones. These findings suggest that the normal function of ß4 integrin as mechanical adhesive device is enhanced upon vimentin downregulation. As a proof of principle, the compromised invasive potential of vimentin depleted cells could be rescued upon blocking with ß4 integrin adhesion-blocking (ASC-8) antibody or downregulation of ß4 integrin in vimentin knockdown background. Interestingly, plectin which associates with α6ß4 integrin in the hemidesmosomes, was also found to be upregulated in vimentin knockdown clones. Furthermore, experiments on lysosome and proteasome inhibition revealed that perhaps vimentin regulates the turnover of ß4 integrin and plectin. Moreover, an inverse association was observed between vimentin expression and ß4 integrin in oral squamous cell carcinoma (OSCC). Collectively, our results show a novel role of vimentin in modulating cell motility by destabilizing ß4 integrin-mediated adhesive interactions. Further, vimentin-ß4 integrin together may prove to be useful markers for prognostication of human oral cancer.

PKD2 and RSK1 Regulate Integrin ß4 Phosphorylation at Threonine 1736.

The integrin α6ß4, a major component of hemidesmosomes (HDs), stabilizes keratinocyte cell adhesion to the epidermal basement membrane through binding to the cytoskeletal linker protein plectin and association with keratin filaments. Disruption of the α6ß4-plectin interaction through phosphorylation of the ß4 subunit results in a reduction in adhesive strength of keratinocytes to laminin-332 and the dissolution of HDs. Previously, we have demonstrated that phosphorylation of T1736 in the C-terminal end of the ß4 cytoplasmic domain disrupts the interaction of ß4 with the plakin domain of plectin. Furthermore, we showed that ß4-T1736 can be phosphorylated by PKD1 in vitro, and although both PMA and EGF induced T1736 phosphorylation, only PMA was able to activate PKD1. Here, we show that depletion of [Ca2+]i augments PMA- and EGF-induced phosphorylation of ß4-T1736 and that this is caused by inhibition of the calcium-sensitive protein phosphatase calcineurin and augmentation of ERK1/2 activation. We also show that in keratinocytes the PMA-stimulated phosphorylation of ß4-T1736 primarily is mediated by PKD2 activation downstream of PKCδ. On the other hand, both the EGF-stimulated phosphorylation of T1736 and the EGF-induced dissolution of HDs are dependent on a functional MAPK signaling pathway, and treatment with the RSK inhibitor BI-D1870 prevented EGF-stimulated phosphorylation of ß4-T1736. Moreover, phosphorylation of ß4-T1736 is enhanced by overexpression of wild-type RSK1, while it is reduced by the expression of kinase-inactive RSK1 or by siRNA-mediated depletion of RSK1. In summary, our data indicate that different stimuli can lead to the phosphorylation of ß4-T1736 by either PKD2 or RSK1.

Morphologic changes of corneal epithelial flap: ethanol-mediated versus mechanical removal.

PURPOSE: To evaluate morphologic changes in human corneal epithelial flap removed mechanically or after ethanol application. METHOD: Epithelial corneal flap was removed after ethanol application (20 eyes) or mechanically (19 eyes). Any changes were studied by transmission electron microscopy. RESULTS: Thirty-nine eyes were enrolled in the study. The following changes were found in the alcohol-applied group: apoptotic cells, membrane-bound blebs with marked dilatation of endoplasmic reticulum, and short intercellular cleavage with approximately one-third of cell length depth. In mechanical debridement, cleavages extended more than half of the cell length by tearing hemidesmosomes. CONCLUSION: Alcohol application leads to cell damage in basal epithelial cells but cleavage plane remains smooth. Generally, none of the methods caused trauma to the basement membrane.

P2Y2 receptor inhibits EGF-induced MAPK pathway to stabilise keratinocyte hemidesmosomes.

α6ß4 integrin is the main component of hemidesmosomes (HD) that stably anchor the epithelium to the underlying basement membrane. Epithelial cell migration requires HD remodelling, which can be promoted by epidermal growth factor (EGF). We previously showed that extracellular nucleotides inhibit growth factor-induced keratinocyte migration. Here, we investigate the effect of extracellular nucleotides on α6ß4 integrin localisation in HD during EGF-induced cell migration. Using a combination of pharmacological inhibition and gene silencing approaches, we found that UTP activates the P2Y2 purinergic receptor and Gαq protein to inhibit EGF/ERK1/2-induced cell migration in keratinocytes. Using a keratinocyte cell line expressing an inducible form of the Raf kinase, we show that UTP inhibits the EGF-induced ERK1/2 pathway activation downstream of Raf. Moreover, we established that ERK1/2 activation by EGF leads to the mobilisation of α6ß4 integrin from HD. Importantly, activation of P2Y2R and Gαq by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of α6ß4 integrin, plectin, BPAG1, BPAG2 and CD151 in keratinocytes. Finally, we demonstrated that the activation of p90RSK, downstream of ERK1/2, is sufficient to promote EGF-mediated HD dismantling and that UTP does not stabilise HD in cells expressing an activated form of p90RSK. Our data underline an unexpected role of P2Y2R and Gαq in the inhibition of the ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration.

Morphological evidences indicate that the interference of cimetidine on the peritubular components is responsible for detachment and apoptosis of Sertoli cells.

Cimetidine, referred as antiandrogenic agent, has caused alterations in the seminiferous tubules, including alterations in the peritubular tissue and death of myoid cells by apoptosis. Regarding the structural and functional importance of the peritubular tissue for the maintenance of Sertoli cells (SC), we purpose to investigate the SC-basement membrane interface, focusing the morphological features of SC and their interaction with the basement membrane in the affected tubules by cimetidine. Ten animals were distributed into two groups, control (CG) and cimetidine (CmG) which received saline solution and 50 mg of cimetidine per kg of body weight, respectively, for 52 days. The testes were fixed, dehydrated and embedded for analyses under light and transmission electron microscopy. Paraffin sections were submitted to the TUNEL method; sections of testes embedded in glycol methacrylate were submitted to PAS method and stained by H&E for morphological and quantitative analyses of Sertoli Cells. In the CmG, the SC nuclei were positive to the TUNEL method and showed typical morphological alterations of cell death by apoptosis (from early to advanced stages). A significant reduction in the number of Sertoli Cells was probably due to death of these cells by apoptosis. A close relationship between SC nuclear alterations (including a high frequency of dislocated nuclei from the basal portion) and damage in the peritubular tissue was observed. The ultrastructural analysis showed a parallelism between the gradual advancement of apoptotic process in SC and detachment of the anchoring sites (hemidesmosomes) of SC plasma membrane from the lamina densa. The presence of portions of lamina densa underlying the detached hemidesmosomes indicates a continuous deposition of lamina densa, resulting in the thickening of the basal lamina. The results indicate a possible disarrangement of the SC cytoskeleton, including the focal adhesion structure. These alterations are related to SC apoptosis and probably result from disturbs induced by cimetidine on the peritubular tissue.

Equine laminitis: ultrastructural lesions detected 24-30 hours after induction with oligofructose.

REASONS FOR PERFORMING STUDY: The pathology of equine laminitis has been well-documented 48 h after dosing with oligofructose when clinical lameness and lamellar disintegration is well advanced. Further analysis of the earliest lesions, by collecting lamellar samples at the first sign of foot lameness after oligofructose dosing is required in order to increase understanding of the disease. OBJECTIVES: To investigate lamellar epidermal hemidesmosome damage and basement membrane dysadhesion by transmission electron microscopy (TEM). METHODS: Eight clinically normal, mature Standardbred horses were divided randomly into 2 groups of 4. The treatment group were dosed with oligofructose (10 g/kg bwt) and subjected to euthanasia when shifting weight from one foot to other commenced and at the first sign of lameness during walking and turning. This occurred at 24 h in 3 horses and 30 h in one. The sham treatment control group were dosed with water and subjected to euthanasia after 48 h. Lamellar tissues of the front feet were harvested and processed for ultrastructural study using TEM. RESULTS: Examination by TEM showed excessive waviness of the basement membrane zone and pointed tips of some secondary epidermal lamellae, an ultrastructural lesion typical of laminitis. The average number of hemidesmosomes/microm of basement membrane was decreased and their distance from the centre of the lamina densa of the basement membrane was increased. CONCLUSIONS: Laminitis lesions are detectable 24 h after oligofructose administration. POTENTIAL RELEVANCE: Hindgut events occurring in the first 24 h after dosing have begun the destruction of the hoof lamellar interface. Prevention and treatment strategies should precede lameness if they are to be efficacious.

Serine phosphorylation of the integrin beta4 subunit is necessary for epidermal growth factor receptor induced hemidesmosome disruption.

Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of alpha6beta4 to plectin plays a central role in their assembly. We have defined three regions on beta4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of beta4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of beta4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the beta4 subunit.

Naltrexone accelerates healing without compromise of adhesion complexes in normal and diabetic corneal epithelium.

Naltrexone (NTX) is an opioid antagonist that accelerates wound healing of corneal epithelium in normal and diabetic animals. Junctional complexes (hemidesmosomes) are important in establishing adhesion of the corneal epithelium to the stroma. This study was designed to examine whether NTX, at a concentration that enhances corneal re-epithelialization, influences the appearance and number of hemidesmosomes in Normal, diabetic (DB) (hyperglycemic), and DB animals receiving insulin (DB-IN) (normoglycemic), and treated topically with NTX (10(-4) M) or sterile vehicle (SV) for 7 days following abrasion. Electron microscopic analysis of the peripheral cornea 2 weeks after removal of the epithelium indicated hemidesmosomes that could be classified into four sectional profiles. No differences were detected in either the structure or the number of junctional complexes in the cornea between Normal, DB, or DB-IN groups receiving vehicle or treated with NTX. Moreover, the fine structure of the basal and suprabasal layers of the corneal epithelium in all groups--including those treated with NTX--were comparable. These results indicate that topical application of NTX accelerates diabetic corneal epithelial healing without causing morphologic abnormalities in the reassembly of adhesion structures. Furthermore, controlled and uncontrolled diabetes for up to 3 months does not affect corneal adhesion complexes when compared to normal corneas. Thus, recurrent erosion following abrasion of the diabetic cornea, with preservation of the basal lamina, cannot be explained by structural abnormalities in the reformation of the epithelial adhesion complex.

The short arm of laminin gamma2 chain of laminin-5 (laminin-332) binds syndecan-1 and regulates cellular adhesion and migration by suppressing phosphorylation of integrin beta4 chain.

The proteolytic processing of laminin-5 at the short arm of the gamma2 chain (gamma2sa) is known to convert this laminin from a cell adhesion type to a motility type. Here, we studied this mechanism by analyzing the functions of gamma2sa. In some immortalized or tumorigenic human cell lines, a recombinant gamma2sa, in either soluble or insoluble (coated) form, promoted the adhesion of these cells to the processed laminin-5 (Pr-LN5), and it suppressed their migration stimulated by serum or epidermal growth factor (EGF). Gamma2sa also suppressed EGF-induced tyrosine phosphorylation of integrin beta4 and resultant disruption of hemidesmosome-like structures in keratinocytes. Gamma2sa bound to syndecan-1, and this binding, as well as its cell adhesion activity, was blocked by heparin. By analyzing the activities of three different gamma2sa fragments, the active site of gamma2sa was localized to the NH(2)-terminal EGF-like sequence (domain V or LEa). Suppression of syndecan-1 expression by the RNA interference effectively blocked the activities of domain V capable of promoting cell adhesion and inhibiting the integrin beta4 phosphorylation. These results demonstrate that domain V of the gamma2 chain negatively regulates the integrin beta4 phosphorylation, probably through a syndecan-1-mediated signaling, leading to enhanced cell adhesion and suppressed cell motility.

Retinoic acid disintegrated desmosomes and hemidesmosomes in stratified oral keratinocytes.

BACKGROUND: Although it is known that retinoic acid (RA) regulates the cellular differentiation of skin keratinocytes, the effects of RA on the anchoring junction have not been clarified. The effects of all-trans RA on cell-cell and cell-matrix connections of gingival epithelial (GE)1 cells in a multilayered culture were investigated. METHODS: Ultrastructures of GE1 cells were observed and immunohistochemistry was used to detect keratin 4, keratin 13, and desmoglein expression. Reverse transcription-polymerase chain reaction was performed to detect expression of desmosome and hemidesmosome-associating adhesion molecules, keratin 13, and keratin14. RESULTS: Retinoic acid caused immunohistochemical diminution of keratin 4, keratin 13, and desmoglein. Ultrastructurally, RA induced drastic loss of typical desmosomes and complete loss of hemidesmosomes. RA significantly decreased the transcript levels of keratin 13, keratin 14, desmoglein 1, and desmocollin 1 in a dose-dependent manner. The 230-kD bullous pemphigoid antigen (BPAG1) gene expression was also reduced by RA, whereas transcript levels of integrin alpha6, integrin beta4, the 180-kD bullous pemphigoid antigen (BPAG2), and laminin 5 were not affected. CONCLUSION: These results indicated that RA disintegrated not only desmosomes by depriving the cells of desmoglein 1, desmocollin 1, keratin 13, and keratin 4, but also hemidesmosomes by reducing the expression of BPAG1 and keratin 14 in basal keratinocytes.

Equine laminitis: loss of hemidesmosomes in hoof secondary epidermal lamellae correlates to dose in an oligofructose induction model: an ultrastructural study.

REASONS FOR PERFORMING STUDY: Light microscopical studies show that the key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. More precise knowledge of the damage occurring in the lamellar basement membrane zone may result if laminitis affected tissue is examined with the transmission electron microscope. This could lead to better understanding of the pathogenesis of lesions and the means of treatment or prevention. OBJECTIVES: To investigate the ultrastructure of acute laminitis as disease of greater severity is induced by increasing oligofructose (OF) dosage. METHODS: Three pairs of normal horses, dosed with OF at 7.5, 10 and 12.5 g/kg bwt via nasogastric intubation, developed laminitis 48 h later. Following euthanasia, their forefeet were processed for transmission electron microscopy. Lamellar basal cell hemidesmosome (HD) numbers and the distance between the basal cell plasmalemma and the lamina densa of the basement membrane were estimated and compared to control tissue. RESULTS: Increasing OF dosage caused greater HD loss and more severe laminitis. The characteristic separation of the basement membrane, cytoskeleton failure and rounded basal cell nuclei results from combined HD dysassembly and anchoring filament failure. CONCLUSIONS: Without properly assembled HDs, dysadhesion between the lamina densa of the basement membrane (BM) and epidermal basal cells occurs, emphasising the fundamental importance of HDs in maintaining attachment at the lamellar interface. Medical conditions that trigger lamellar matrix metalloproteinase (MMP) activation and/or compromise entry of glucose into lamellar basal cells appear to promote loss and failure of HDs and, therefore, laminitis development. POTENTIAL RELEVANCE: A correlation between lameness severity and escalating loss of lamellar HDs now exists. Therapy aimed at protecting the lamellar environment from haematogenous delivery of MMP activators or from glucose deprivation may control laminitis development.

A dynamic podosome-like structure of epithelial cells.

Focal contacts and hemidesmosomes are cell-matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin, and zyxin. We also show that the maintenance of the actin core and hemidesmosomes is dependent on actin polymerization, src-family kinases, and Grb2, but not on microtubules. Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of beta4 integrin subunit to the actin core before its positioning at hemidesmosomes. When 804G cells are induced to migrate, actin cores as well as hemidesmosomes disappear and beta4 integrin subunit becomes co-localized with dynamic actin at leading edges. We show that podosome-like structures are not unique to cells of mesenchymal origin, but also appear in epithelial cells, where they seem to be related to basement membrane adhesion.

Tyrosine phosphorylation: a critical component in the formation of hemidesmosomes.

Our goal was to evaluate the role of tyrosine phosphorylation in the complete formation of hemidesmosomes that occurs during development or during remodeling after injury. A corneal organ culture system was used to study hemidesmosome formation as it would occur in an intact tissue. Phosphorylation of the integrin subunit beta 4 and bullous pemphigoid antigen-1 (BPAG-1) was examined, as these proteins are known to play a role in linking the electron-dense plaques along the basal surface with the intermediate filaments to complete the formation of hemidesmosomes. Corneal epithelial sheets were placed on substrata that contained an intact basal lamina or basal laminae that had been either modified or removed. These constructs were incubated for up to 18 h, and hemidesmosome formation was evaluated by using transmission electron microscopy. When epithelial sheets were placed on intact basal laminae and incubated in the presence of the tyrosine kinase inhibitor genistein (200 microM), hemidesmosome formation was impaired. The formation of electron-dense regions was delayed, and no association of intermediate filaments was detected. Results were confirmed by biochemical studies. When the epithelium and underlying proteins were extracted and immunoprecipitated with beta 4 or BPAG-1, tyrosine phosphorylation decreased in the presence of genistein. In addition, the phosphorylation of beta 4 decreased when epithelial sheets were incubated on substrata from which the basal lamina had been removed or altered. Thus, a reduction in phosphorylation of tyrosine residues impairs the formation of mature hemidesmosomes, and substrata that fail to support hemidesmosome formation also demonstrate decreased phosphorylation of tyrosine residues.

Role of vitamins C and E as chemopreventive agents in the hamster cheek pouch treated with the oral carcinogen-DMBA.

OBJECTIVE: To evaluate the role of vitamins C and E as chemopreventive agents in oral carcinogenesis by optical and ultrastructural studies. MATERIALS AND METHODS: The cheek pouch of male hamsters was treated with the oral carcinogen, dimethylbenz(a)anthracene (DMBA), to induce multiple tumour formation. Vitamins C and E were applied either singly or in combination as a chemopreventive agent. Paraffin and resin-embedded sections of the hamster cheek pouch were studied optically and ultrastructurally. RESULTS: The epithelium of control hamsters showed hyperorthokeratosis and parakeratosis, but did not develop well differentiated squamous cell carcinoma (WDSCC). Ninety percent of the animals treated with DMBA alone showed WDSCC while 10% of the animals developed papillomas. There was also a marked increase in the number of cells undergoing mitosis in this group. A reduction in the yield (1.1 tumour/animal) and rate 60-80% of squamous cell carcinomas but not of papillomas (2.0 papillomas/animal) was observed in groups VI-VIII treated with DMBA and vitamins C and E singly or in combination as compared to those of DMBA only. In animals treated with DMBA plus vitamins C and E, statistical significant decrease in the number of animals with tumours and mitotic basal cells was observed when compared with the DMBA treated group. Control animals showed normal ultrastructural morphology while tumour-bearing animals showed basal lamina in a discontinuous, fragmented, broken and diffused basement membrane, with diminished lamina densa, fewer hemidesmosomes and invagination of the basal cell cytoplasmic processes in the subepithelium. CONCLUSION: These results indicate that vitamin E singly or in combination with vitamin C plays a role in the inhibition of tumour cell growth.

Effect of short chain fatty acids on human gingival epithelial cell keratins in vitro.

Hemidesmosomal attachment of the junctional epithelial cells to the tooth and the ability of the attached cells to divide are essential features of the healthy dentogingival junction. Short chain fatty acids are bacterial metabolites associated with gingival inflammation and periodontal pockets. In vitro, short chain fatty acids have been shown to inhibit epithelial cell division and increase the density of their keratin filaments. This study examined these keratin changes by making use of human gingival keratinocyte cultures, gel electrophoresis and Western blot. Short chain fatty acids, butyrate and propionate, increased the relative amount of keratin proteins in the cells, most strikingly keratin K17. The distribution of K17 was further studied in a culture model for human junctional epithelium and in gingival biopsies. In butyrate-treated cultures of junctional epithelium, K17 expression was markedly increased and extended to the basal cells and to the cells mediating the attachment of the explant to the substratum. In clinically healthy gingiva, K17 was expressed predominantly in sulcular epithelium. The dividing basal cells and the cells attached to the tooth were negative. In advanced periodontitis, a strong reaction for K17 was localised to the pocket epithelium. The inhibition of epithelial cell division and the simultaneous upregulation of K17 in vitro, and the strong expression of this protein in detached pocket epithelium suggest a role for the short chain fatty acids in the degenerative process that leads to subgingival advancement of pathogens and, eventually, to periodontal pocket formation.