Human epidermal keratinocytes are a source of tenascin-C during wound healing.

Tenascin-C is a large hexameric extracellular matrix glycoprotein that is expressed in a temporally and spatially restricted pattern associated with stromal-epithelial interactions. In adult human skin, the expression level of tenascin-C is low, but tenascin-C is abundantly present in the dermal compartment during embryogenesis and wound healing and in skin tumors. Herein we have investigated the cellular source of tenascin-C production in human skin, both in vivo and in vitro, by using immunohistochemistry, mRNA in situ hybridization, western blotting, and an enzyme-linked immunosorbent assay. In addition we studied the cell-matrix interaction between epidermal keratinocytes and purified tenascin-C. By using in vitro culture models, we found that keratinocytes not only synthesize and secrete tenascin-C but can also deposit tenascin-C in de-epidermized dermis in a pattern that is very similar to that in vivo. In vivo, during wound healing of normal human skin, we found tenascin-C extracellularly in the wound bed and also in a granular pattern within the neo-epidermis. By mRNA in situ hybridization, we could identify the basal migrated keratinocytes as the main source of tenascin-C in the early phase of wound healing. In the granulation phase, tenascin-C expression by the keratinocytes is downregulated. Cultured keratinocytes were found to adhere poorly to tenascin-C, and those that did adhere retained a rounded morphology. We conclude that human keratinocytes are a major source of tenascin-C during the early phase of wound healing, and we hypothesize that tenascin-C is unlikely to be an adhesive substrate for migrating keratinocytes.

Tenascin-C expression in human epidermal keratinocytes is regulated by inflammatory cytokines and a stress response pathway.

Recently we showed that human epidermal keratinocytes express the extracellular matrix protein tenascin-C (TN-C) during wound healing, but not in normal adult skin. To gain further insight into the regulation of epidermal TN-C expression, we tested the effect of various stimuli on TN-C expression by cultured keratinocytes. Our results indicate that IL-4 is a very strong inducer of TN-C protein and mRNA expression in normal keratinocytes. Furthermore, TNFalpha and IFNgamma moderately increased TN-C expression. No other cytokines and growth factors that we tested, including various factors that stimulate TN-C expression in mesenchymal cells, significantly affected TN-C secretion by cultured keratinocytes. The regulation of TN-C expression in keratinocytes is distinct from that of fibronectin, since IL-4 and IFNgamma did not affect fibronectin expression in our experiments, and TNFalpha only slightly increased fibronectin levels. To investigate the role of cellular stress response pathways that can be activated by TNFalpha in the regulation of TN-C expression, we tested the effect of different inhibitors and an activator of these intracellular signalling cascades. The results show that the p38 MAP-kinase pathway is not involved in TNFalpha-induced TN-C expression in cultured keratinocytes. Activation of the JNK/SAPK-1 pathway by the addition of sphingomyelinase resulted in a dose-dependent increase of TN-C expression. TN-C expression by squamous carcinoma cell lines was differentially affected by the cytokines that stimulated TN-C expression in normal keratinocytes: TNFalpha again increased TN-C secretion, but IL-4 and IFNgamma had little effect. We conclude that there are distinct regulation mechanisms for TN-C expression in normal keratinocytes, tumor-derived keratinocytes and mesenchymal cells. The observation that TN-C is abundant in inflamed skin is a strong indication that inflammatory cytokines such as IL-4, TNFalpha and IFNgamma could also be involved in the regulation of epidermal TN-C expression in vivo.

Tenascin-C expression in prostatic intraepithelial neoplasia (PIN): a marker of progression?

BACKGROUND: Tenascin (tenascin-C) has been suggested to be associated with active epithelial-stromal interactions. We evaluated tenascin expression in tissue remodelling processes presumably associated with PIN and prostate carcinoma (PCa). MATERIALS AND METHODS: Tenascin immunoreactivity was evaluated in 38 PIN lesions (low-grade = 5, high-grade = 33) from 27 paraffin-embedded PCa specimens, and compared with expression in pre-existent (normal) prostate, benign prostatic hyperplasia (BPH), and PCa. RESULTS: Periepithelial stromal tenascin expression was low in low-grade PIN, and similar to normal glands and BPH, whereas expression in high-grade PIN was high and partly overlapped that of well-/moderately differentiated PCa. High-grade PCa usually expressed little, if any tenascin. CONCLUSIONS: The variable periglandular tenascin expression in high-grade PIN may reflect the biologic behaviour of this lesion, and may be indicative of variable levels of tissue remodelling. In well/moderately differentiated PCa tenascin expression levels may be an indicator of tumour progression.

Expression of tenascin-C splice variants by human skin cells.

Tenascin-C is an extracellular matrix glycoprotein that is expressed in a spatially and temporally restricted pattern. Various functionally different tenascin-C isoforms can be expressed as a result of alternative splicing of the pre-mRNA. Previously we identified human epidermal keratinocytes as a source of tenascin-C in healing wounds. In this study, we investigated whether different tenascin-C transcripts are expressed by epidermal keratinocytes and dermal fibroblasts. In addition, we compared expression of tenascin-C splice variants at the mRNA and protein levels in tissue samples of normal and diseased skin. Northern blot analysis revealed two major tenascin-C mRNA transcripts of approximately 7500 and 5800 nucleotides in cultured epidermal keratinocytes and fibroblasts, and in biopsies. Although both dermal fibroblasts and epidermal keratinocytes predominantly expressed the larger tenascin-C mRNA, epidermal keratinocytes expressed smaller transcripts at higher levels than dermal fibroblasts. In keratinocytes the levels of the two mRNAs were differentially affected by inflammatory cytokines that increased tenascin-C expression in these cells. The addition of IFN gamma slightly increased the proportion of large transcripts. In contrast, TNF alpha favoured expression of smaller tenascin-C transcripts, and IL-4 equally affected the expression of large and small tenascin-C mRNAs. To enable detection of tenascin-C transcripts that are expressed at very low levels, we amplified by polymerase chain reaction the fibronectin type III repeats whose expression is regulated by alternative splicing. In cDNA of cultured keratinocytes and fibroblasts, and in skin biopsies, several tenascin-C transcripts could be detected that corresponded to tenascin-C variants including different numbers of fibronectin type III repeats. Distribution of tenascin-C isoforms at the protein level was studied immunohistochemically in healthy skin, wounds, psoriatic lesions and epidermal tumours and hyperplasia. No differences were observed in reactivity between an antibody that binds all tenascin-C isoforms and antibodies that bind fibronectin type III repeats that can be spliced out from smaller tenascin-C isoforms. We conclude that the tenascin-C isoforms that are translated from transcripts that we identified at the mRNA level seem to be distributed similarly in the conditions investigated.

Epicutaneous application of leukotriene B4 induces patterns of tenascin and a heparan sulfate proteoglycan epitope that are typical for psoriatic lesions.

Application of leukotriene B4 (LTB4) to normal human skin induces changes similar to those found in psoriatic skin, and it has proved to be a useful model for studying the pathogenesis and treatment of psoriasis. We studied the expression patterns of molecules that have recently been shown to be altered in lesional psoriatic skin, including the extracellular matrix protein tenascin (TN) and the basement membrane and cell surface-associated heparan sulfate proteoglycans (HSPGs). During 72-h the expression of these markers was studied immunohistochemically and the expression of TN was correlated with epidermal proliferation and influx of inflammatory cells. Following the peak influx of polymorphonuclear leukocytes, a marked increase in TN expression was noted in the papillary dermis 72 h after LTB4 application. The expression patterns of basal membrane-associated epitopes of HSPG remained unaltered, whereas the expression of cell surface-associated HSPG disappeared 72 h after LTB4 application. A significant correlation was found between dermal TN expression and epidermal hyperproliferation, and between TN expression and the presence of dermal T cells. These findings indicate that the model of LTB4-induced acute cutaneous inflammation displays many characteristics of early psoriatic lesions and could serve as a model to study some of the cell biological changes in this disease.

Tenascin-C degradation in chronic wounds is dependent on serine proteinase activity.

Degradation of extracellular matrix (ECM) components by proteinases is part of the physiological remodelling process during normal wound healing. Excessive degradation of the ECM, however, is likely to create an environment that can no longer support keratinocyte migration and is thought to play a role in the impaired healing of chronic ulcers. Tenascin-C is an ECM component that is markedly upregulated in acute and chronic wounds. Here we report on our investigations into the degradation of tenascin-C in chronic venous leg ulcers. We found proteolytic fragments of tenascin-C in leg ulcer exudate. We also detected fragments of fibronectin in the wound fluid and in addition observed breakdown of fibronectin by wound fluid in vitro, as has previously been reported by others. Wound fluid of four out of six chronic leg ulcers degraded purified human tenascin-C in vitro, and degradation of tenascin-C correlated with high levels of functionally active leucocyte elastase and metalloproteinases in the wound fluid. To identify which proteinases were involved in tenascin-C degradation, we tested the effect of specific proteinase inhibitors. The addition of EDTA or E64 did not protect tenascin-C from degradation, suggesting that neither metalloproteinases nor cysteine proteinases are responsible for cleavage. Tenascin-C breakdown was inhibited by PMSF and SKALP/elafin, and we therefore conclude that leucocyte elastase and possibly other serine proteinases are the tenascin-C-degrading enzymes in ulcer exudate. Taking into account the possible effects of tenascin-C and tenascin-C fragments on cell behaviour, we hypothesize that degradation of tenascin-C could affect the healing process in chronic venous ulcers.

Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to translocate proteins across both membranes of the cell envelope. Many of these proteins are transported via the type II secretion pathway and adopt their tertiary conformation in the periplasm, which implies the presence of a large transport channel in the outer membrane. The outer membrane protein, XcpQ, which is involved in transport of folded proteins across the outer membrane of P. aeruginosa, was purified as a highly stable homomultimer. Insertion and deletion mutagenesis of xcpQ revealed that the C-terminal part of XcpQ is sufficient for the formation of the multimer. However, linker insertions in the N-terminal part can disturb complex formation completely. Furthermore, complex formation is strictly correlated with lethality, caused by overexpression of xcpQ. Electron microscopic evaluation of the XcpQ multimers revealed large, ring-shaped structures with an apparent central cavity of 95 A. Purified PilQ, a homologue of XcpQ involved in the biogenesis of type IV pili, formed similar structures. However, the apparent cavity formed by PilQ was somewhat smaller, 53 A. The size of this cavity could allow for the transport of intact type IV pili.

Test of the combinatorial model of intron recognition in a native maize gene.

Previous studies have established that splice site selection and splicing efficiency in plants depend strongly on local compositional contrast consisting of high exon G+C content relative to high intron U content. The combinatorial model of plant intron recognition posits that splice site sequences as well as local intron and exon sequences contribute to splice site selection and splicing efficiency. Most of the previous studies used synthetic or chimeric constructs, often tested in heterologous hosts. To perform a more critical test of the combinatorial model in a native context, the single intron of the maize Bronze2 gene and its flanking exons were modified by site-directed mutagenesis. Splicing efficiency was tested in maize protoplasts. Results show that a higher U content in the flanking 5' exon, whether close to or distant from the 5' splice site, did not modify splicing efficiency. Decreasing exon G+C content dramatically impaired splicing. Increasing intron G+C content or decreasing intron U content adversely impacted splicing. In all constructs splicing occurred exclusively at the original 5' and 3' splice sites. These results are consistent with the hypothesis that exon G+C content and intron U content contribute separate but complementary aspects of intron definition in the native Bz2 transcript.

Tenascin expression during wound healing in human skin.

In adult human skin, the expression of the extracellular matrix glycoprotein tenascin is limited. Under hyperproliferative conditions such as psoriasis and epidermal tumours, dermal tenascin expression is strongly upregulated. The aim of this study was to investigate the pattern and kinetics of tenascin expression in human skin during wound healing and to address the question of whether keratinocytes can directly interact with tenascin during re-epithelialization. Tenascin expression was investigated in excisional wounds in normal human skin, in explants of normal human skin, and in chronic venous ulcers, using immunohistochemistry. No tenascin staining was found directly underneath the leading edge of the sheet of migrating keratinocytes in the excisional wounds and explants. In the excisional wounds and the ulcers, dermal tenascin was strongly upregulated in areas adjacent to hyperproliferative epidermis. These hyperproliferative areas are located approximately 10-50 cells behind the leading edge, as assessed by staining for the Ki-67 antigen and the proliferating cell nuclear antigen (PCNA). At the later stages of normal wound healing and in the chronic ulcers, tenascin was also detected in the wound bed. In these areas, the dermal-epidermal junction stained positive for laminin but was negative for heparan sulphate. The absence of the latter basement membrane component suggests that the formation of a new basement membrane is not completed in these wounds. These findings suggest that tenascin is not a substrate for migrating keratinocytes; that the rapid induction of tenascin expression in the papillary dermis during wound healing results from interaction with the hyperproliferative epidermis; and that in the later stages of wound healing, keratinocytes can potentially interact with tenascin in the wound bed, because the basement membrane of the neo-epidermis is incomplete.

Basal membrane heparan sulphate proteoglycan expression during wound healing in human skin.

Heparan sulphate proteoglycans (HSPGs) are integral components of the basement membrane (BM) in various tissues. HSPGs are important in the assembly and structure of the BM, and their putative functions include regulation of basement membrane permeability, binding of growth factors, and a role in cellular adhesion. In this study the expression of HSPG was examined during wound healing in human skin, using monoclonal antibodies (MAbs) that recognize the HSPG core protein and two different heparan sulphate (HS) epitopes, and the dynamics of HSPG expression were investigated in relation to epidermal cellular proliferation and permeability of the BM. Healing of excisional wounds in healthy volunteers was studied from day 0 up to 1 year. Intact human skin showed strong continuous staining of the dermo-epidermal BM and the vascular BM with all MAbs. Up to day 4 after wounding, staining for HSPG was absent under the ingrowing epidermis, with any of the MAbs, indicating that no complete BM was present. From day 7 onwards, the BM of the neo-epidermis showed positive staining for the HSPG core protein and a low sulphated HS epitope, and after day 14, the staining intensity was similar to normal skin. The staining patterns of these HSPG epitopes were similar to that of laminin. The staining pattern with a MAb against an epitope in the highly sulphated part of HS was found to be distinct from the other BM markers studied. This epitope was absent under the neo-epidermis up to 2 months after wounding. One year after wounding, the epitope was found to be present again. We observed that only in the time period between 2 months and 1 year had the epidermis normalized with respect to the number of cycling cells and the absence of high molecular weight plasma proteins. These findings suggest a correlation between normalization of epidermal proliferation, BM permeability, and regeneration of BM HS. It is proposed that complete BM maturation following skin wounding is a slow process and may account for the epidermal abnormalities that persist for a considerable period of time after wound healing.

Tenascin-C is not a useful marker for disease activity in psoriasis.

Tenascin-C is an extracellular matrix glycoprotein that is markedly upregulated in the dermis of psoriatic skin. In this study, we have addressed the question whether the presence of tenascin-C in the lesion or in serum is a marker for disease activity. Immunohistochemical staining of tenascin-C before and after treatment with different topical and systemic medication showed that tenascin-C remained abundant after clinical remission of lesions, indicating that downregulation of tenascin-C to normal values is a slow process. By using a sensitive enzyme-linked immunosorbent assay to measure levels of serum tenascin-C in psoriatic patients and unaffected individuals, we found that tenascin-C levels in most patients were within the normal range. Moreover, tenascin-C values did not correlate with disease activity. We conclude that tenascin-C is not useful as a marker for disease activity in psoriasis.

Expression of periglandular tenascin-C and basement membrane laminin in normal prostate, benign prostatic hyperplasia and prostate carcinoma.

OBJECTIVE: To evaluate the structural relationship of the distribution between tenascin (tenascin-C, an extra-cellular matrix glycoprotein involved in stromal-epithelial interactions in both normal and pathological conditions) and laminin, an important component of the basement membrane, in normal and neoplastic human prostate, and to establish whether changes in the basement membrane are accompanied by changes in tenascin staining. MATERIALS AND METHODS: Seventy-five snap-frozen prostate samples representing normal glands, nodular benign prostatic hyperplasia and prostate carcinoma were stained for tenascin. From these, 15 samples were selected for dual-immunofluorescence staining and a confocal laser scan microscope was used to simultaneously visualize tenascin and laminin immunoreactivity. RESULTS: Tenascin was expressed in the extracellular matrix, mainly at the periphery of the glands, in tumour foci and blood vessels. In cases with intact basement membranes, e.g. normal glands and hyperplastic lesions, tenascin expression was weak. Low- and moderate-grade tumours were characterized by strong tenascin expression, while laminin expression was weak and/or showed discontinuities, indicating disturbances in basement membrane composition. High-grade tumours had sparse tenascin staining and a marked loss of laminin immunoreactivity. CONCLUSION: These results indicate that periglandular tenascin expression correlates with the integrity of the basement membrane in the human prostate. By influencing stromal-epithelial interactions, tenascin may play a role in maintaining tissue homeostasis in the prostate.

Epidermal cell kinetics by combining in situ hybridization and immunohistochemistry.

Double labelling can serve as a useful tool for providing information about cell kinetics in normal and hyperproliferative tissues in general, and skin in particular. We have developed a double-labelling method that combines immunohistochemistry using the monoclonal antibody MIB1 and non-isotopic in situ hybridization using either a digoxigenin-labelled RNA probe specific for histone 3 mRNA sequences or a Fluorescein-labelled oligonucleotide probe specific for histone 2b, 3, 4 mRNA sequences. Double labelling was performed on normal, tape-stripped normal skin and psoriatic skin. The three proliferation markers were also examined by single labelling. The ratio of cells in the S-phase (Ns) and the growth fraction (Ncy) was determined. In normal skin, psoriatic skin and tape-stripped normal skin after 24 h and after 48 h, we calculated that 15%, 16%, 3% and 12% of growth fraction consisted of cells in the S-phase respectively. The S-phase lasts approximately 10 h, so the cell cycle time in normal and psoriatic skin is approximately 62.5 h. At present, the MIB1/H3 digoxigenin or MIB1/H2b-H3-H4 Fluorescein double-labelling technique cannot be used routinely. Therefore, in order to understand the cell kinetic processes better, experiments are recommended to optimize these methods. From a practical point of view and for reasons of specificity and sensitivity, we prefer the Fluorescein-labelled oligonucleotide probe method.

Transient tenascin enhancement is an early event after androgen ablation in rat prostate.

Tenascin (tenascin-C), a mesenchymal glycoprotein, is expressed in many tissue remodeling processes. We evaluated tenascin expression during androgen-deprivation-related involution of the rat prostate. At set intervals following castration and subsequent testosterone repletion, prostates were removed in 30 adult rats. Each prostate was immunostained with a polyclonal antiserum against rat tenascin and keratin antibodies specifically directed against exocrine basal cells and luminal cells in the prostate glandular structure. Morphologic impressions were semiquantatively evaluated using a computer-assisted image analysis system. Rat prostates showed a transient increase in the periglandular tenascin expression directly following castration that reached a maximum at day 3. At day 6, tenascin expression was similar to control prostates. This was accompanied by a decrease of cells in the luminal cell layer. The weakest tenascin immunoreactivity was noted on day 14 after androgen withdrawal. This process was reversed by androgen repletion. This study shows that in the rat prostate tenascin expression may be androgen dependent and that during androgen deprivation-related involution tenascin expression is probably associated with tissue remodeling by stromal-epithelial interactions.