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T Kartasova

Publications and source records attributed to T Kartasova.

At least 19 recordsLinked to original sources
PubMed2000

Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein.

The epidermal cornified cell envelope (CE) is a complex protein-lipid composite that replaces the plasma membrane of terminally differentiated keratinocytes. This lamellar structure is essential for the barrier function of the skin and has the ability to prevent the loss of water and ions and to protect from environmental hazards. The major protein of the epidermal CE is loricrin, contributing approximately 70% by mass. We have generated mice that are deficient for this protein. These mice showed a delay in the formation of the skin barrier in embryonic development. At birth, homozygous mutant mice weighed less than control littermates and showed skin abnormalities, such as congenital erythroderma with a shiny, translucent skin. Tape stripping experiments suggested that the stratum corneum stability was reduced in newborn Lor(-/-) mice compared with wild-type controls. Isolated mutant CEs were more easily fragmented by sonication in vitro, indicating a greater susceptibility to mechanical stress. Nevertheless, we did not detect impaired epidermal barrier function in these mice. Surprisingly, the skin phenotype disappeared 4-5 d after birth. At least one of the compensatory mechanisms preventing a more severe skin phenotype in newborn Lor(-/-) mice is an increase in the expression of other CE components, such as SPRRP2D and SPRRP2H, members of the family of "small proline rich proteins", and repetin, a member of the "fused gene" subgroup of the S100 gene family.

Adaptation, Biological↗
PubMed1999

Transglutaminase cross-linking properties of the small proline-rich 1 family of cornified cell envelope proteins. Integration with loricrin.

Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no alpha or beta structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.

Amino Acid Sequence↗
PubMed1999

Mouse Sprr2 genes: a clustered family of genes showing differential expression in epithelial tissues.

Small proline-rich (SPR) proteins are structural components of the cornified cell envelope of stratified squamous epithelia. They are subdivided into three families, i.e., SPR1, SPR2, and SPR3, of which the SPR2 family is the most complex. To understand the significance of this complexity, we have isolated 11 mouse Sprr2 genes, constructed a provisional physical map of the Sprr2 locus on mouse Chromosome 3, and examined the expression patterns of the Sprr2 genes in mouse epithelial tissues. The 11 Sprr2 sequences are highly conserved with a central domain containing a variable number of repeats. In situ hybridization showed the Sprr2 expression to be confined to epithelia. RT-PCR using primers specific for each of the 11 Sprr2 members demonstrated varying degrees of expression among the individual Sprr2 members in different tissues. The correlation between the physical location of the genes in the Sprr2 locus and their expression patterns suggests multiple levels of controlled expression.

Animals↗
PubMed1999

Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis.

The Ku protein is a complex of two subunits, Ku70 and Ku80. Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells. In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G(2)/M phase, which does not require DNA-PKcs.

Antigens, Nuclear↗
PubMed1998

Structural and transglutaminase substrate properties of the small proline-rich 2 family of cornified cell envelope proteins.

The small proline-rich (SPR) proteins are components of the cornified cell envelope of stratified squamous epithelia and become cross-linked to other proteins by transglutaminases (TGases). The SPR2 family is the most complex, as it consists of several differentially expressed members of the same size. To explore their physical and cross-linking properties, we have expressed in bacteria a human SPR2 family member, and purified it to homogeneity. By circular dichroism, it possesses no alpha or beta structure but has some organized structure associated with the central peptide repeat domain. The TGase 1, 2, and 3 enzymes expressed in epithelia use the recombinant SPR2 protein as a complete substrate in vitro, but with widely differing kinetic efficiencies, and in different ways. With TGase 1, only one glutamine on the head domain and one lysine on the tail domain were used for limited interchain cross-linking. With TGase 3, multiple head and tail domain residues were used for extensive interchain cross-linking. The total usage of glutamine and lysine residues in vitro by TGase 3 was similar to that seen in earlier in vivo studies. We conclude that SPR2 proteins are cross-linked in epithelia primarily by the TGase 3 enzyme, a minor extent by TGase 1, and probably not by TGase 2.

Amino Acid Sequence↗
PubMed1998

Biochemical evidence that small proline-rich proteins and trichohyalin function in epithelia by modulation of the biomechanical properties of their cornified cell envelopes.

The cornified cell envelope (CE) is a specialized structure involved in barrier function in stratified squamous epithelia, and is assembled by transglutaminase cross-linking of several proteins. Murine forestomach epithelium undergoes particularly rigorous mechanical trauma, and these CEs contain the highest known content of small proline-rich proteins (SPRs). Sequencing analyses of these CEs revealed that SPRs function as cross-bridgers by joining other proteins by use of multiple adjacent glutamines and lysines on only the amino and carboxyl termini and in functionally non-polar ways. Forestomach CEs also use trichohyalin as a novel cross-bridging protein. We performed mathematical modeling of amino acid compositions of the CEs of mouse and human epidermis of different body sites. Although the sum of loricrin + SPRs was conserved, the amount of SPRs varied in relation to the presumed physical requirements of the tissues. Our data suggest that SPRs could serve as modifiers of a composite CE material composed of mostly loricrin; we propose that increasing amounts of cross-bridging SPRs modify the structure of the CE, just as cross-linking proteins strengthen other types of tissues. In this way, different epithelia may use varying amounts of the cross-bridging SPRs to alter the biomechanical properties of the tissue in accordance with specific physical requirements and functions.

Amino Acid Sequence↗
PubMed1998

Small proline-rich proteins are cross-bridging proteins in the cornified cell envelopes of stratified squamous epithelia.

The cornified cell envelope (CE) is a specialized structure which contributes barrier function to stratified squamous epithelial cells. It is composed of an amalgam of several structural proteins that are rendered insoluble by isopeptide bond crosslinking by transglutaminases. One set of the structural proteins present in CEs of most such epithelia are the small proline rich (SPR) proteins, which are a family of about 12 related structural proteins. We have recovered a large number of peptides containing isopeptide crosslinks, including 236 involving SPR proteins, following proteolysis of CEs isolated from foreskin epidermal tissue and cultured epidermal keratinocytes. Analysis of this database has provided novel information on their function. First, we found that SPRs became crosslinked to many other structural proteins within the CE. Second, multiple glutamine and lysine residues located only on the amino- and carboxy-termini of the SPR proteins were involved in crosslinking, so that the two ends are functionally equivalent. Third, the SPRs functioned as cross-bridging proteins, by directly adjoining other CE structural proteins. In the specialized case of the epidermal CE, the SPRs cross-bridged between loricrin. In cultured keratinocytes which make little loricrin and serve as a model for internal stratified squamous epithelia, the SPRs formed extensive cross-bridges among themselves. Thus SPRs are ubiquitous cross-bridging proteins whose differential expression patterns apparently reflect specific barrier requirements of different epithelia.

Animals↗
PubMed1997

Involucrin and SPRR are synthesized sequentially in differentiating cultured epidermal cells.

Epidermal keratinocytes form cornified cell envelopes during terminal differentiation. These envelopes are composed of several cross-linked molecules, including involucrin, loricrin, and SPRR. We have previously reported that involucrin is synthesized earlier in terminal differentiation than loricrin. To further elucidate the mechanisms of terminal differentiation, we have now examined the expression of the two differentiation markers, involucrin and SPRR, in cultured human epidermal keratinocytes. In confluent nonstratified cultures, many involucrin-immunoreactive cells were detected, but few SPRR1/3-positive cells. Double staining demonstrated that cells containing SPRR1/3 almost always contained involucrin, but involucrin was present in many cells that did not contain SPRR. Light and electron microscopic immunohistochemistry of a stratified culture demonstrated that lower cells (close to the basal layer) were occasionally involucrin-positive, but lacked SPRR1/3, whereas more superficial cells contained both involucrin and SPRR. We conclude that involucrin and SPRR are sequentially induced in this order during keratinocyte differentiation.

Adult↗
PubMed1996

DMSO induces apoptosis in SV40-transformed human keratinocytes, but not in normal keratinocytes.

We found that dimethyl-sulfoxide (DMSO) at concentrations of 2.5% induced apoptosis in SV40-immortalized human keratinocytes, while normal keratinocytes were arrested at the boundary of G1/S phase under the same conditions. DMSO-induced apoptosis in SV-40 immortalized keratinocytes was not associated with change in phosphorylated state of the retinoblastoma susceptibility gene. When SV40-immortalized cells were treated with 2.5% DMSO, dissociation of the complex was observed by immunoblotting of SV40 T antigen from immunoprecipitated p53 protein fraction.

Antigens, Viral, Tumor↗
PubMed1996

Sequence and expression patterns of mouse SPR1: Correlation of expression with epithelial function.

A final event in the terminal differentiation of stratified squamous epithelia is the formation of a cornified cell envelope, which is a complex of several proteins cross-linked together by transglutaminases. One set of proteins is the family of small proline rich (SPR) proteins. In human foreskin epidermal cell envelopes, SPRs serve as cross-bridging proteins among the more abundant loricrin. In order to study further their evolution and expression, we have isolated and sequenced cDNAs encoding two mouse SPR1 proteins, SPR1a and SPR1b Comparative sequence analysis showed the preservation of the overall structure of mammalian SPR1 proteins with highly conserved termini and a central peptide domain repeated 13 (SPE1a) or seven (SPR1b) times. Tissues obtained from mouse fetal, newborn, and adult skin were tested by Northern blot analyses, in situ hybridization and immunohistochemistry using an antibody raised to a synthetic peptide corresponding to the C terminus of the SPR1a protein. Skin expression was first detected in fetal periderm in anagen hair follicles of newborn and older mice, and in the thickened epidermis of the lip and footpad, but no signal was detected in interfollicular trunk epidermis. High levels of SPR1a expression were found in epithelia from the forestomach and penis, and in benign squamous papillomas. Other epithelia expressing SPR1a include the tongue, esophagus, and vagina. Whenever detected, SPR1a positive staining was present in the spinous and granular layers. In the forestomach and papillomas, the periphery of cells in the cornified layer was also stained. Our results suggest that SPR1a participates widely in the construction of cell envelopes in cornifying epithelia characterized by either increased thickness or a requirement for extreme flexibility. Based on its likely function as a cross-bridging protein in cell envelopes, we conclude that the mechanical attributes of cell envelopes may be determined in part by the SPR1 content, in accordance with the specific function of the epithelium.

Animals↗
PubMed1996

Differentiation-associated localization of small proline-rich protein in normal and diseased human skin.

The expression of SPRR (small proline-rich protein) was investigated in normal human skin and in diseased skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma, naevus pigmentosus, ichthyosis vulgaris and several inflammatory skin diseases, by immunohistochemical staining. A polyclonal antibody was raised against a synthetic peptide for a C-terminal common region for SPRR1 and SPRR3. In immunoblot analysis, a positive band of 18 kDa was detected, which showed the presence of SPRR1 in human epidermal keratinocytes. In normal epidermis, positive staining for SPRR was observed in keratinocytes in the granular layer and the uppermost or two spinous cell layers, with no staining of the other spinous or basal layers. The staining was obvious at the cell periphery, weak at the cytoplasm, and absent in the nucleus. Staining was observed in several outer layers of the follicular infundibulum to the isthmus. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, eccrine duct, melanocytes, Langerhans cells or fibroblasts. The arrectores pilorum, striated muscles, muscle layers of vessels, and myoepithelia of eccrine gland, were weakly stained. In psoriatic skin, stained keratinocytes were distributed in the spinous cell layers except for the basal layer. In ichthyosis vulgaris, SPRR was barely expressed in the uppermost living cell layers of the epidermis. In epidermolytic hyperkeratosis, degenerated squamous cells widely expressed SPRR. In Darier's disease, dyskeratotic cells were clearly stained. In squamous cell carcinoma, staining was observed in keratotic cells around horny pearls. In basal cell epithelioma, naevus pigmentosus, and malignant melanoma, the tumour cells or naevus cells were not stained. The distribution of SPRR was similar to that of involucrin in normal and several diseased skin, except for ichthyosis vulgaris. We conclude that SPRR is expressed in close association with epidermal differentiation in normal skin and skin diseases. The alteration of the expression of the proteins correlated to terminal differentiation, and differs from disease to disease.

Adult↗
PubMed1996

Differential expression and cell envelope incorporation of small proline-rich protein 1 in different cornified epithelia.

In the final stages of terminal differentiation in the epidermis and other squamous epithelia, a approximately 15 nm thick protein layer called the cornified cell envelope (CE) assembles on the keratinocytes' inner surface. Its constituent proteins are covalently crosslinked by the action of transglutaminases. Recent studies have indicated that the expression of CE precursor proteins may vary in different tissues. To investigate such variations further, we have studied the CEs of two different keratinizing epithelia of mouse: epidermis and forestomach, with particular focus on their contents of loricrin and the small proline-rich proteins (SPRs). To this end, we have applied electron microscopic immunocytochemistry and estimated the CE protein compositions by mathematical modeling of their amino acid compositions. Ultrastructurally, forestomach resembles the epidermis in having well defined cornified and granular layers. Minor but significant differences are: in forestomach, striated material resembling lamellar granules is intercalated between the cornified squames; and in forestomach granular layer cells, loricrin-containing L-granules are more abundant, and filaggrin-containing F-granules less abundant than in epidermis. In forestomach, dense labeling with anti-SPR1 antibody was observed at the margin of cornified layer cells; and in the granular layer, diffuse but positive labeling of both cytoplasm and nucleus. In contrast, epidermis was uniformly negative. Isolated forestomach CEs (but not epidermal CEs), labeled positively on the cytoplasmic side, consistent with the presence of covalently crosslinked SPR1. Our compositional analysis predicts the content of loricrin in forestomach CEs to be very high (approximately 65%), as in the epidermis, and accompanied by approximately 18% content of total SPRs. Of these, a substantial proportion should be SPR1, according to our immunolabeling data. In contrast, epidermal CEs are calculated to have a much lower amount of SPRs or SPR-like proteins (approximately 8%), with a negligible content of SPR1. Thus both kinds of CEs have loricrin as their major constituent but differ in their respective complements of SPRs, which are thought to inter-connect loricrin molecules in the final phase of CE assembly. Applying a basic concept of materials science, it may be that the observed differences in their SPR contents reflect differences in the mechanical and chemical properties required for the function of the respective CEs.

Amino Acid Sequence↗
PubMed1995

Altered distribution of keratinization markers in epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (EH) is a genetic disorder of keratins associated with epidermal differentiation. Affected individuals carry gene mutations for conserved sequences of keratins K1 or K10. The structural alterations of tonofilaments in EH seem to be a direct consequence of the keratin gene mutations. EH epidermis, however, shows many other unexplained abnormalities including acanthosis, hypergranulosis, and hyperkeratosis. To further elucidate the pathogenetic mechanism of EH, we studied distribution patterns of other keratinization-associated molecules including involucrin, small proline-rich protein (SPRR) 1, loricrin and trichohyalin in the skin of four patients by light and electron microscopic immunohistochemistry in conjunction with conventional transmission electron microscopy. The middle to upper epidermal cells showed moderate to strong immunoreactivities to involucrin, SPRR1 and loricrin antibodies. Both intracellular staining and cell peripheral staining was seen for involucrin and SPRR1 antibodies. Loricrin labelling was prematurely associated with the plasma membrane of granular cells, possibly relating to abnormal keratin filament aggregation and cellular vacuolization. Some loricrin labelling was localized on the keratin aggregates, suggesting intermolecular associations between keratin and loricrin. Trichohyalin, hardly detectable in normal epidermis, was present in some granular and cornified cells in EH in association with keratin filaments, suggesting that it may function as an intermediate filament-associated protein. While cornified cell envelopes were intensely labelled only with loricrin antibodies in normal skin, they were immunoreactive to involucrin, SPRR1 and loricrin antibodies in EH. Sequential change in electron density of the cornified cell envelopes, a constant feature in normal skin, was often absent in EH. These results suggest an altered assembly process of cornified cell envelopes in EH.

Adolescent↗
PubMed1993

Hyperphosphorylation of keratins by treatment with okadaic acid of BALB/MK-2 mouse keratinocytes.

Protein hyper- or hypophosphorylation induced by okadaic acid (OA) treatment was examined using quiescent cultures of the BALB/MK-2 mouse epidermal keratinocytes. Treatment with OA enhanced the phosphorylation of five proteins with molecular weights of 65,000, 55,000, 50,000, 28,000 and 15,000 (p65, p55, p50, p28, and p15, respectively) and decreased that of two proteins with molecular weights of 22,000 and 20,000 (p22 and p20, respectively). The two major phosphorylated proteins, p65 and p55, were identified as type II and type I keratins, respectively, by immunoblotting and immunoprecipitation with keratin specific antibodies. Serine was the only phosphoamino acid residue in hydrolysates of the 32P-labeled keratins purified from OA-treated cells. Two-dimensional tryptic peptide maps of the phosphorylated keratins showed that the hyperphosphorylation was largely due to phosphorylation at several additional sites in both keratins. The hyperphosphorylation of keratins induced by OA treatment resulted in a drastic change in their solubility. This change closely correlated with reorganization of the keratin filament network, which finally collapsed into large perinuclear aggregates. Concomitantly the cells changed from a typical epithelial shape to a round shape. Of several protein kinase inhibitors tested, only staurosporine interfered with this OA-induced morphological change and reorganization of the keratin network.

Animals↗
PubMed1993

Mouse differentiation-specific keratins 1 and 10 require a preexisting keratin scaffold to form a filament network.

Keratins 1 (K1) and 10 (K10) are the predominant cytoskeletal intermediate filaments of epidermal cells during transition from the proliferative to the terminal differentiation stage. In situ, formation of the K1/K10 intermediate filament network occurs in the cytoplasm of cells with a preexisting cytoskeleton composed of keratins 5 and 14. To define cytoskeletal interactions permissive for formation of the K1/K10 filamentous network, active copies of mouse K1 and K10 genes were introduced into fibroblasts (NIH 3T3) which do not normally express these proteins. Transient and stable transfectants, as well as heterokaryons produced by fusions with epithelial cells, were evaluated for expression of K1 and K10 proteins and filament formation using specific antibodies. In contrast to keratin pairs K5/K14 and K8/K18, the K1/K10 pair failed to form an extensive keratin filament network on its own, although small isolated dense K1/K10 filament bundles were observed throughout the cytoplasm by EM. K1 and K10 filaments integrated only into the preexisting K5/K14 network upon fusion of the NIH 3T3 (K1/K10) cells with epithelial cells expressing endogenous K5/K14 or with NIH 3T3 cells which were transfected with active copies of the K5 and K14 genes. When combinations of active recombinant gene constructs for keratins 1, 5, 10, and 14 were tested in transient NIH 3T3 transfections, the most intact cytokeratin network observed by immunofluorescence was formed by the K5/K14 pair. The K1/K14 pair was capable of forming a cytoskeletal network, but the network was poorly developed, and usually perinuclear. Transfection of K10 in combination with K5 or K1 resulted in cytoplasmic agglomerates, but not a cytoskeleton. These results suggest that the formation of the suprabasal cytoskeleton in epidermis is dependent on the preexisting basal cell intermediate filament network. Furthermore, restrictions on filament formation appear to be more stringent for K10 than for K1.

3T3 Cells↗
PubMed1992

Relationship between the expression of differentiation-specific keratins 1 and 10 and cell proliferation in epidermal tumors.

In normal epidermis, the expression of keratins 1 and 10 is associated with the loss of proliferative capacity and the onset of terminal differentiation. Keratins 1 (K1) and 10 (K10) are commonly expressed in the differentiating layer of benign tumors, but are lost during progression from the benign to the malignant state in skin carcinogenesis. Active gene constructs of mouse K1 and K10 were introduced into papilloma and carcinoma cell lines derived from keratinocytes to analyze the consequences of the expression of these keratins on the organization of the endogenous cytoskeletal network and on the mitotic activity of the recipient cells. Exogenous K1 integrated into the preexisting keratin K5/K14 network of both SLC-1 carcinoma and 308 papilloma cells. The formation of a recombinant cytoskeleton was more restricted for K10 than for K1 and appeared to be related to a requirement for cessation of cell division before K10 could integrate. The integration of exogenous K1 filaments into the endogenous keratin network was compatible with sustained proliferation of SLC-1 carcinoma cells in vitro. However, the exogenous gene was not expressed in tumor grafts in vivo. In contrast, stable K1 or K10 transfectants could not be selected in 308 cells, suggesting that benign tumor cells expressing suprabasal keratins cannot sustain proliferation.

Animals↗