Novel transglutaminase-1 mutations and genotype-phenotype investigations of 104 patients with autosomal recessive congenital ichthyosis in the USA.

BACKGROUND: Autosomal recessive congenital ichthyosis (ARCI) is a rare hereditary disorder of cornification. Mutations in the transglutaminase-1 (TGM1) gene, which encodes for the epidermal enzyme transglutaminase-1 (TGase-1), are one of the causes of ARCI. METHODS: The TGM1 mutation spectrum was characterised and genotype-phenotype correlations investigated in 104 patients with ARCI ascertained through the National Registry for Ichthyosis and Related Disorders in the USA. Methods: Germline mutations in TGM1 were identified in 55% (57/104) of patients with ARCI. Arginine residues in TGase-1 were mutated in 39% (22/57) of patients overall and 54% (20/37) of those with missense mutations. In total, 55% (12/22) of missense mutations were within CpG dinucleotides and 92% (11/12) of these mutations were C-->T or G-->A transitions. The genotype-phenotype investigation found that ARCI with TGM1 mutations was significantly associated with presence of collodion membrane at birth (p = 0.006), ectropion (p = 0.001), plate-like scales (p = 0.005) and alopecia (p = 0.001). Patients who had at least one mutation predicted to truncate TGase-1 were more likely to have more severe hypohidrosis (p = 0.001) and overheating (p = 0.0007) at onset of symptoms than were those with exclusively TGM1 missense mutations. A logistic model was developed, which predicted that individuals with collodion membrane, alopecia and/or eye problems are about four times more likely to have TGM1 mutations than patients without these findings. CONCLUSION: This is the largest investigation of patients with ARCI to date. It expands the TGM1 mutation spectrum and confirms that despite genetic and phenotypic heterogeneity in ARCI, TGM1 is the main causative gene for this disorder. The high frequency of mutated arginine codons in TGM1 may be due to the deamination of CpG dinucleotides.

Differentiation of cultured keratinocytes promotes the adherence of Streptococcus pyogenes.

Based on a consideration of the histopathology of nonbullous impetigo that shows localization of Streptococcus pyogenes to highly differentiated, subcorneal keratinocytes, we hypothesized that adherence of an impetigo strain of S. pyogenes would be promoted by terminal differentiation of keratinocytes. An assay was developed in which S. pyogenes adhered via pilus-like projections from the cell wall to the surface of cultured human keratinocytes in a time- and inoculum-dependent manner suggestive of a receptor-mediated process. Terminal differentiation of keratinocytes was induced by increasing the calcium concentration in the growth medium, and was confirmed by morphologic analysis using electron microscopy. Adherence of S. pyogenes was three and fourfold greater to keratinocytes differentiated in 1.0 and 1.5 mM calcium, respectively, compared with undifferentiated keratinocytes in 0.15 mM calcium. The presence of calcium during the adherence assay further enhanced adherence nearly twofold. Adherence occurred preferentially to sites of contact between adjacent keratinocytes, suggesting that the keratinocyte receptor may be a molecule involved in cell-to-cell adhesion. In contrast, nonpathogenic Streptococcus gordonii adhered poorly to keratinocytes regardless of their state of terminal differentiation, and adherence of a pharyngeal strain of S. pyogenes was twofold greater to undifferentiated than differentiated keratinocytes. This is the first report of in vitro adherence of S. pyogenes to keratinocytes in a manner that emulates human impetigo. Adherence of only the impetigo strain, and not the pharyngeal strain of S. pyogenes or the nonpathogenic S. gorgonii isolate, was promoted by keratinocyte differentiation. This result provides a model system for investigating the molecular pathogenesis of streptococcal skin infections.

A mouse model to evaluate the interface between skin and a percutaneous device.

Percutaneous medical devices are integral in the management and treatment of disease. The space created between the skin and the device becomes a haven for bacterial invasion and biofilm formation and results in infection. We hypothesize that sealing this space via integration of the skin into the device will create a barrier against bacterial invasion. The purpose of this study was to develop an animal model in which the interaction between skin and biomaterials can be evaluated. Porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] rods were implanted for 7 days in the dorsal skin of C57 BL/6 mice. The porous poly(HEMA) rods were surface-modified with carbonyldiimidazole (CDI) or CDI plus laminin 5; unmodified rods served as control. Implant sites were sealed with 2-octyl cyanoacrylate; corn pads and adhesive dressings were tested for stabilization of implants. All rods remained intact for the duration of the study. There was histological evidence of both epidermal and dermal integration into all poly(HEMA) rods regardless of treatment. This in vivo model permits examination of the implant/skin interface and will be useful for future studies designed to facilitate skin cell attachment where percutaneous devices penetrate the skin.

Inducible expression of filaggrin increases keratinocyte susceptibility to apoptotic cell death.

Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. We have previously shown that filaggrin constructs, when transiently transfected into epithelial cells, lead to a collapsed keratin cytoskeletal network and dysmorphic nuclei with features of apoptosis. The apparent transfection rate is low with filaggrin constructs, supporting their disruptive role but hindering further study. To bypass this problem, we generated stable keratinocyte cell lines that express mature human filaggrin using a tetracycline-inducible promoter system. We found that cell lines expressing filaggrin, but not control cell lines, exhibited increased sensitivity to multiple apoptotic stimuli as measured by morphologic and biochemical criteria. None of the cell lines showed an increase in endogenous expression of filaggrin in response to the same stimuli. Filaggrin expression alone was insufficient to induce apoptosis in these keratinocyte cell lines. We conclude that filaggrin, due to its keratin binding ability, primes cells for apoptosis. Because filaggrin is expressed at a level of the epidermis where keratinocytes are in transition between the nucleated granular and the anucleate cornified layers, we hypothesize that filaggrin aids in the terminal differentiation process by facilitating apoptotic machinery.

Epidermal growth factor stimulates ornithine decarboxylase activity in cultured mammalian keratinocytes.

The influence of epidermal growth factor (EGF) on ornithine decarboxylase has been examined in cultured bovine keratinocytes. Keratinocyte ornithine decarboxylase activity was maximal at pH 6.3 in MES buffer in the presence of dithiothreitol and EDTA. When cultured cells, deprived of serum, were exposed to EGF, the activity of ornithine decarboxylase was stimulated severalfold. Enzyme activity increased in a dose-dependent manner with EGF. The time course of this stimulation is unlike any previously reported in cultured cells. The increase in activity was maximal by 8 h. A small dip in activity was seen between 8 and 12 h. Increased activity was sustained for as long as 24 h after exposure to EGF. The prolonged increased in enzyme activity was reduced by actinomycin D. When cycloheximide was added 1 h before EGF, ornithine decarboxylase activity was obliterated. This is the first demonstration of ornithine decarboxylase stimulation following exposure to EGF in cultured keratinocytes. The prolonged duration of ornithine decarboxylase stimulation is unexplained but may be related to processing of EGF by the keratinocytes.

Retinoic acid regulates oral epithelial differentiation by two mechanisms.

The effect of retinoic acid (RA) concentration on differentiation of oral keratinocytes and the influence of fibroblasts on RA-dependent regulation were investigated in a lifted culture system. Keratinocyte differentiation was assessed by morphology, immunohistochemistry and immunoblotting. Filaggrin/profilaggrin and keratin 1 were used as biochemical markers for cornified epithelium and keratins 13 and 19 as markers for noncornified epithelium. Cultured oral keratinocytes in RA-free conditions differentiated in a manner that closely resembled the differentiation pattern of gingival epithelia in vivo. Increasing RA concentrations altered the in vivo-like terminal differentiation of oral keratinocytes by disruption of organized stratification, inhibition of filaggrin/profilaggrin and K1 expression, and stimulation of K13 and K19 expression. Differentiation of keratinocytes from both cornified and noncornified regions of the oral cavity varied in a similar manner in response to added RA, with the exception of K19 expression. K19 was consistently expressed at higher levels in keratinocytes originating from noncornified epithelial as compared to those from cornified epithelia. The level of RA regulation was ultimately dependent on the type of fibroblasts underlying the epithelial cells. Homologous fibroblasts rendered the oral keratinocytes less sensitive to the effects of RA than skin fibroblasts. In addition, at a given RA concentration, fibroblasts from cornified oral mucosa potentiated keratinocyte expression of RA sensitive markers of keratinization as compared to the influence exerted by fibroblasts originating from noncornified oral mucosa. These results indicate that the RA regulation of oral epithelial differentiation is mediated by two separate mechanisms: a direct, RA concentration-dependent effect, and an indirect, fibroblast-mediated effect.

Retinoic acid regulates oral epithelial differentiation by two mechanisms.

The effect of retinoic acid (RA) concentration on differentiation of oral keratinocytes and the influence of fibroblasts on RA-dependent regulation were investigated in a lifted culture system. Keratinocyte differentiation was assessed by morphology, immunohistochemistry and immunoblotting. Filaggrin/profilaggrin and keratin 1 were used as biochemical markers for cornified epithelium and keratins 13 and 19 as markers for noncornified epithelium. Cultured oral keratinocytes in RA-free conditions differentiated in a manner that closely resembled the differentiation pattern of gingival epithelia in vivo. Increasing RA concentrations altered the in vivo-like terminal differentiation of oral keratinocytes by disruption of organized stratification, inhibition of filaggrin/profilaggrin and K1 expression, and stimulation of K13 and K19 expression. Differentiation of keratinocytes from both cornified and noncornified regions of the oral cavity varied in a similar manner in response to added RA, with the exception of K19 expression. K19 was consistently expressed at higher levels in keratinocytes originating from noncornified epithelia as compared to those from cornified epithelia. The level of RA regulation was ultimately dependent on the type of fibroblasts underlying the epithelial cells. Homologous fibroblasts rendered the oral keratinocytes less sensitive to the effects of RA than skin fibroblasts. In addition, at a given RA concentration, fibroblasts from cornified oral mucosa potentiated keratinocyte expression of RA sensitive markers of keratinization as compared to the influence exerted by fibroblasts originating from noncornified oral mucosa. These results indicate that the RA regulation of oral epithelial differentiation is mediated by two separate mechanisms: a direct, RA concentration-dependent effect, and an indirect, fibroblast-mediated effect.

Harlequin ichthyosis keratinocytes in lifted culture differentiate poorly by morphologic and biochemical criteria.

Harlequin ichthyosis (HI) is a severe congenital ichthyosis in which massively thickened stratum corneum with abnormal barrier function often results in death of affected newborns. Survivors evolve into a severe nonbullous ichthyosiform erythroderma. Previously we have ascertained three biochemical phenotypes of HI, based on abnormal profilaggrin and K6 and K16 expression in epidermis. Submerged cultures of HI keratinocytes differentiated abnormally, but the three phenotypes were indistinguishable in vitro. We hypothesized that differentiation in submerged culture was insufficient to reflect in vivo biochemical abnormalities or that dermal components might be necessary for expression. To test these hypotheses HI keratinocytes and fibroblasts (n = 3) were grown on collagen gels at the air-medium interface in a cross-over design with normal keratinocytes and fibroblasts. Epithelia derived from lifted cultures were studied by light microscopy and immunocytochemistry and extracted for western blot analysis. In contrast to our prediction, lifted cultures of HI keratinocytes formed a poorly differentiated epithelium, and normal keratinocytes formed an epidermal-like tissue with expression of K1 and expression and processing of profilaggrin to filaggrin. In addition, the presence of HI fibroblasts consistently altered differentiation of both HI and normal keratinocytes, resulting in less complete morphologic differentiation. The findings suggest that both epithelial and mesenchymal elements of the skin from HI are affected but that the primary abnormality lies in the keratinocytes.

Reduced stability and bi-allelic, coequal expression of profilaggrin mRNA in keratinocytes cultured from subjects with ichthyosis vulgaris.

Ichthyosis vulgaris (IV) is an inherited scaling skin disorder in which expression of profilaggrin is reduced. Previous studies have indicated that the reduction is caused by defective post-transcriptional control of gene expression. Here we present evidence that profilaggrin mRNA in keratinocytes cultured from subjects with IV is intrinsically unstable and has a shorter half-life compared with that in normal cells. When IV-affected keratinocytes were treated with the protein synthesis inhibitor cycloheximide, the steady-state level of profilaggrin mRNA was increased due to stabilization of the transcript. In addition, the number of filaggrin repeats within the profilaggrin gene was studied. The number of filaggrin repeats (10-12) in individuals with IV did not differ from that of unaffected subjects. Expression of the gene was bi-allelic and coequal in both control and affected individuals. Our results suggest a model in which a labile ribonuclease and a stabilizing factor may modulate the profilaggrin mRNA steady-state level in normal cells, whereas the stabilizing factor may be absent or functionally inactive in IV-affected keratinocytes.

Long-term culture of murine epidermal keratinocytes.

The production of transgenic and null mice with skin abnormalities makes it increasingly important to establish cultures of mouse epidermal keratinocytes for in vitro studies. This requires that each cell line be derived from a single mouse and that the cells be carried for multiple passages. Freezing the cells would also be advantageous by allowing comparison of keratinocytes from several mouse lines at the same time. Mouse keratinocytes, however, have been exceedingly difficult to grow as primary cultures, and subculturing these cells has been virtually impossible until now. We describe a gentle dissociation method and a highly supplemented fibroblast conditioned medium that allows us to grow and subculture total mouse keratinocytes for up to 19 subcultures, allowing an increase in cell number of greater than 10 logs. Epidermal keratinocytes from newborn mice were grown on collagen IV coated dishes in murine fibroblast conditioned medium with 0.06 mM calcium and added growth factors. The cells could be passaged, frozen as viable stocks, and induced to differentiate. Morphologically the cultured keratinocytes demonstrated a pattern characteristic of basal cells. Stratified cultures which made mouse keratin 1 and profilaggrin through passage 10 were induced by purging the monolayer cultures of growth factors, then adding medium with 0.15 mM calcium; expression of mouse keratin 1 and profilaggrin was lost by passage 15. The methods explained in detail here should be of great interest to investigators who are now trying to analyze skin phenotypes and expression of markers of epidermal differentiation of their transgenic or knockout mice.

Profilaggrin, a high-molecular-weight precursor of filaggrin in human epidermis and cultured keratinocytes.

Filaggrin is a histidine-rich matrix protein of keratinized epidermis. Filaggrin is synthesized as a high-Mr, phosphorylated precursor, profilaggrin, that is processed to form the lower-Mr product present in cornified cells. This study reports the identification of profilaggrin in human epidermis and in unusually well-differentiated cultured human keratinocytes with the use of a polyclonal antihuman filaggrin antiserum. Polyclonal antiserum raised against human filaggrin stained keratohyaline granules and stratum corneum in tissue sections of human skin. Analysis of epidermal extracts showed an immunoreactive low-Mr band (37,000), previously identified as filaggrin, and an immunoreactive high-Mr band (greater than 220,000). Both [32P] phosphate and [3H]histidine were incorporated into the high-Mr band after pulse labeling for 3 h. After a 15-h chase, [3H]histidine, but not [32P]phosphate appeared in filaggrin. Human foreskin keratinocytes cultured on a 3T3 feeder layer were unusually well differentiated. Numerous well-formed keratohyaline granules, complete desmosomes, lamellar granules, and cornified cell envelopes were observed. Immunofluorescence with antihuman filaggrin antiserum showed a granular staining pattern in the more stratified cells. Extracts of cultured cells contained a diffuse high-Mr immunoreactive band but no immunoreactive equivalent of filaggrin. These studies suggest that human skin filaggrin, like rodent filaggrin, is synthesized as a high-Mr, phosphorylated, histidine-rich precursor (profilaggrin) that is processed via posttranslational modification to filaggrin. In human keratinocyte cultures a similar high-Mr precursor is present, but evidence of processing to the lower-Mr product, filaggrin, is lacking.

Glycosaminoglycan synthesis by proliferating and differentiated human keratinocytes in culture.

Glycosaminoglycan (GAG) synthesis and compartmentalization were studied in populations of human neonatal keratinocytes under conditions of proliferation and terminal differentiation in vitro. Following isotopic labeling with the precursors [3H]glucosamine and [35S]sulfate, GAGs were extracted from the keratinocytes into several operationally created compartments associated with the cells and extracellular matrix. Chondroitin sulfate and heparan sulfate accounted for the majority of the incorporated label in all preparations. Although total sulfated GAGs per culture increased from proliferative to differentiated conditions, GAG content normalized to the DNA content of the cultures demonstrated the reverse trend. This was particularly evident for the chondroitin sulfates, which declined 60-70% in the differentiated cultures. Furthermore, label incorporation into chondroitin and heparan sulfates revealed a relative compartmental shift to a trypsin-accessible site upon keratinocyte differentiation. An analysis of heparan sulfate structure by characterization of the oligosaccharide products resulting from low pH nitrous acid deaminative degradation provided evidence that the parent material from differentiated keratinocytes contains a larger region of N-sulfated glucosamine residues unassociated with ester sulfate groups. The correlation of variations in GAG content and compartmentalization with the growth condition of human keratinocytes constitutes evidence that this heterogeneous group of cell surface-associated carbohydrates is involved in some aspect of cell function associated with growth control or differentiation. Furthermore, the apparent differences in heparan sulfate primary structure indicate that there is structure-function specificity to this association.

Glycosaminoglycan synthesis by human keratinocytes: cell growth and medium calcium effects.

The influences of cell density, differentiation, and medium calcium levels on glycosaminoglycan biosynthesis were evaluated in cultured human epidermal keratinocytes. Following metabolic labeling with [35S]-sulfate and [3H]-glucosamine under steady state conditions in "high" medium calcium (greater than 1.0 mMol), the majority of sulfated glycosaminoglycans remained associated with the cell layers, whereas hyaluronic acid, which was present in smaller amounts than the sulfated products, was about equally distributed between the medium and the cell layers. Of the sulfated glycosaminoglycans, heparan sulfate and chondroitin 4/6-sulfate were the major species and were present in roughly comparable amounts, whereas dermatan sulfate was quantitatively the lesser of the products. The effects of "low" medium calcium (0.3 and 0.025 mM) were complex, although a consistent decrease in the incorporation of the [3H]-glucosamine precursor was found at high cell density, probably reflecting a decrease in its intracellular specific activity. In "high" calcium cultures, there was a strong inverse correlation (r = -0.92) between keratinocyte cell number and cellular production of sulfated glycosaminoglycans, whereas no such relationship was evident in cultures grown in "low" calcium medium at comparable cell density. Because keratinocyte differentiation is inhibited in the low calcium conditions, the results suggest that the decrease in production of sulfated glycosaminoglycans by confluent keratinocytes may actually correlate with differentiation rather than with cell number.

Keratinocytes cultured from subjects with ichthyosis vulgaris are phenotypically abnormal.

Ichthyosis vulgaris (IV) is an autosomal dominant, scaling disorder in which keratohyaline granules and filaggrin are reduced in or absent from the epidermis of affected individuals. Morphologic and biochemical markers of epidermal differentiation were studied in keratinocytes cultured from clinically unaffected skin of patients with IV, from clinically unaffected skin of an obligate gene carrier, and from normal skin of unaffected family members and an adult volunteer. Cultured keratinocytes from affected subjects formed thickened layers of scaly cells that failed to react with monoclonal antibody to filaggrin. In contrast, normal cells contained many large, immunoreactive granules. Electron microscopy confirmed the absence of keratohyaline granules in affected cells and the presence of large keratohyaline granules in normal cells. Immunoblot analysis of keratinocyte extracts from subjects with ichthyosis showed that profilaggrin was absent, but no differences in keratins were detected between affected and control cells. For all parameters, findings in cells of the clinically unaffected obligate gene carrier were intermediate between those from affected patients and controls. We conclude that keratinocytes cultured from patients with IV maintain structural and biochemical phenotypic characteristics of the disease in vitro.

The distinctive pattern of proteoglycan and glycosaminoglycan free chain synthesis by cultured human epidermal keratinocytes.

The in vitro synthesis of proteoglycans and glycosaminoglycan free chains was studied in human epidermal keratinocytes. Preconfluent and confluent cultures established on 3T3 feeders were steady state labeled with [35S]-sulfate and [3H]-glucosamine after removal of the 3T3 cells. Products in nonionic detergent extracts of keratinocytes and in the medium were analyzed in the presence of protease inhibitors. Glycosaminoglycans as proteoglycans and as free chains were defined by susceptibility or resistance, respectively, to alkaline borohydride reduction. Products associated with the cells were approximately 30% proteoglycans and approximately 70% glycosaminoglycan free chains, whereas in the medium virtually all was proteoglycan. The heparan and chondroitin sulfate proteoglycans were small compared to those of many other cell types. Their Kav on Sepharose CL-4B was 0.56 (estimated 50 kDa), whereas the free chain Kav was 0.74 (estimated 12 kDa). Relative amounts of the sulfated products varied with confluence and differentiation; heparan and chondroitin sulfates were equally represented within the free chains and proteoglycans of the cells in preconfluent, proliferating cultures, whereas in postconfluent, differentiated cultures the major labeling was in the heparan sulfate products, consistent with our prior reports (J Invest Dermatol 88:215-9, 1987 and 91:492-8, 1988). The cellular localization of the products was probed with glycosaminoglycan degrading enzymes added to isotopically prelabeled cultures. The proteoglycans appeared to be located on the external surface of plasma membranes, whereas the glycosaminoglycan free chains resisted digestion and are either intracellular or membrane associated, but otherwise inaccessible. These data establish the distinctive pattern of low Mr proteoglycans and abundant cell-associated glycosaminoglycan free chains synthesized by keratinocytes.

Characterization of two monoclonal antibodies to human epidermal keratohyalin: reactivity with filaggrin and related proteins.

Two monoclonal antibodies (AKH1 and AKH2) were elicited with partially purified human filaggrin and characterized by immunohistochemistry on normal and abnormal skin biopsies, immunoblotting techniques, and antigen purification. Both antibodies react strongly with the granular cell layer consistent with the distribution of keratohyalin and show a more diffuse reaction with the stratum corneum in normal skin biopsies. Reaction in cultured human keratinocytes is limited to immunofluorescent granules in flattened, well-differentiated cells in confluent cultures, in which we have previously demonstrated keratohyalin. On immunoblots AKH1 reacts with filaggrin (37 kD) and profilaggrin (400 kD), while AKH2 primarily stains bands of 150 and 300 kD. The AKH2 antigens were identified in the cationic protein fraction used for immunization and were purified by gel permeation and high-performance liquid chromatography. Amino acid composition of these proteins differs only slightly from filaggrin. Immunohistochemical staining patterns of the two antibodies are very similar in the genetic disorders of keratinization tested, except for ichthyosis vulgaris, and reflect the presence and distribution of keratohyalin. In ichthyosis vulgaris, AKH1 staining is weak, consistent with the morphology and with biochemical absence of profilaggrin/filaggrin; however, AKH2 staining is positive, although weaker than normal, suggesting the presence of the AKH2 antigens even when keratohyalin is absent or abnormal. Antibodies AKH1 and AKH2 may be useful as differentiation markers for keratinization in tissues and for cells in culture. Antibody AKH1 can be used specifically for detection of profilaggrin/filaggrin in tissues, cultured keratinocytes, and extracts.

CHILD syndrome: lack of expression of epidermal differentiation markers in lesional ichthyotic skin.

Congenital hemidysplasia with ichthyosiform erythroderma and limb defects (CHILD) syndrome is a rare genetic disorder. The epidermal abnormalities associated with the unilateral ichthyosis have previously been examined only by morphology. In order to describe these abnormalities more completely we analyzed the expression of markers of epidermal differentiation (keratins and filaggrin), grew keratinocytes in culture, and correlated the results with ultrastructural features. Expression of all differentiation markers was significantly reduced or absent, whereas keratins K5 and K14 and keratins K6 and K16 were strongly expressed in lesional epidermis, suggesting that basal cell keratin expression was not down-regulated as in normal epidermis and that lesional keratinocytes mature via an abnormal pathway. When removed from the tissue and grown in culture, keratinocytes from lesional and non-lesional biopsies had similar phase microscopic morphology as well as keratin and profilaggrin expression, in contrast to the extreme differences in vivo. Lesional keratinocytes also had similar contents of keratin filaments and keratohyalin, but showed abnormal accumulation of intercellular vesicles and debris and altered cell-cell and cell-substratum interaction. Comparison of the results in tissue and in culture suggests that systemic or dermal factors influence the abnormal structural protein expression and ichthyosiform epidermal differentiation seen in CHILD syndrome, but that lesional keratinocytes maintain abnormalities in the secretion and accumulation of extracellular material in vitro similar to the lesional tissue in vivo.

Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity.

Filaggrin is an intermediate filament-associated protein (IFAP) that aggregates epidermal keratin filaments in vitro and is thought to perform a similar function during terminal differentiation in vivo. To test this function in living cells, we transiently expressed constructs encoding human filaggrin in both simple epithelial cells (COS-7) and rat keratinocytes. Scanning laser confocal microscopy showed that filaggrin-positive cells had collapsed keratin and vimentin intermediate filament (IF) networks, and that filaggrin partially co-localized with the IF networks. Filaggrin was also detected diffusely in the cytoplasm and nucleus. In contrast, when profilaggrin-like constructs, containing five filaggrin domains separated by the linker sequences, were expressed in cultured cells, immunoreactive granules formed. This finding is reminiscent of the insoluble nature of native profilaggrin that accumulates in keratohyalin granules in vivo, suggesting that the linker peptides (present in profilaggrin but not filaggrin) are important for granule formation. Cells expressing filaggrin also displayed disruption of the nucleus and the nuclear envelope; they rounded up and lost attachment to the substratum, in contrast to control cells over-expressing beta-galactosidase. This functional test of filaggrin in living cells supports its role in the reorganization and packing of keratin IF in epidermal differentiation. Moreover, the observed effects on cell morphology and nuclear integrity suggest that filaggrin may contribute to the form of apoptosis associated with terminal differentiation in epidermis.

Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: variable morphology and structural protein expression and a defect in lamellar granules.

Skin biopsies and scale samples from nine infants and one fetus affected with harlequin ichthyosis (HI) were obtained from eight families. Epidermal differentiation was examined by morphologic and biochemical criteria and cell culture studies. Two striking abnormalities were identified; first, keratin and filaggrin expression were abnormal and varied between cases, and, second, in all cases lamellar granules were absent or abnormal, and intercellular lamellae within the stratum corneum were absent. Three HI phenotypes were distinguished by variable expression of epidermal structural proteins. Cases were classified by the absence (type 1) or presence (types 2 and 3) of keratins K6 and K16 ("hyperproliferative" keratins) and by the presence of profilaggrin in the interfollicular epidermis (types 1 and 2 only). Profilaggrin is apparently not converted to filaggrin, but it is retained in the scale. The block in profilaggrin processing may be due to an inactive phosphatase. Siblings in two families (presenting with types 1 and 2) showed the same type classification suggesting that expression of the phenotype is consistent within families but differs between families. Cultured HI keratinocytes were normal by phase microscopy, but abnormal by electron microscopy with no lamellar granules and extensive stacking of the upper layers. We conclude that harlequin ichthyosis is a genetically heterogeneous group of disorders with altered lamellar granules, intercellular lipids, and variation in expression and/or processing of structural protein markers of normal epidermal keratinization. Furthermore, the lamellar granule and structural protein defects may be indirectly related via a mechanism involving phosphorylation/dephosphorylation.

Antisense profilaggrin RNA delays and decreases profilaggrin expression and alters in vitro differentiation of rat epidermal keratinocytes.

Ichthyosis vulgaris is an epidermal disorder in which profilaggrin expression is decreased or absent. To determine whether the ichthyosis vulgaris phenotype could be mimicked by eliminating profilaggrin expression, a rat epidermal cell line was transfected with a plasmid that directs the constitutive expression of an RNA that is antisense to normal profilaggrin mRNA. Non-transfected and neomycin-resistant cells not containing antisense plasmid that were grown in the neomycin analogue G418 served as controls. Immunoblot and immunofluorescence analysis showed that profilaggrin protein expression and processing to filaggrin were delayed by 3 to 4 d and decreased in transfected cells. Profilaggrin mRNA was detected in both control and transfected cells only after the cells reached confluence, whereas antisense RNA was detected in transfectants at all times, even prior to confluence. Ultrastructural examination revealed that keratohyalin granules were decreased in number, globular, and heterogeneous in appearance in transfected cells in-contrast to angular structures seen in control cells. Unexpectedly, stratification was impaired, intermediate filaments were noticeably reduced, and cornified cell envelope formation was delayed in transfectants. Unlike ichthyosis vulgaris keratinocytes, where keratin expression is unaffected, appearance of K1 and K10 was delayed and K1/K10 synthesis was delayed and decreased in transfected cells. The precipitous drop in 35S-methionine incorporation into cytoskeletal protein seen at confluence in control cells was delayed by 3 d in transfected cells. We conclude that, rather than producing the ichthyosis vulgaris phenotype, antisense profilaggrin RNA has more broad-reaching effects on in vitro differentiation of rat epidermal keratinocytes.