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H J Stark

Publications and source records attributed to H J Stark.

At least 19 recordsLinked to original sources
PubMed2005

The effects of Aspergillus fumigatus challenge on exhaled and nasal NO levels.

Several studies have previously shown that exposure to indoor air microbes from moisture-damaged buildings can cause adverse health effects. Aspergillus fumigatus is one of the best-documented moulds causing health problems to those exposed. In this study, inhalation of a commercial A. fumigatus solution was assessed, to establish if it would have effects on fractional exhaled (FeNO) and nasal (FnNO) nitric oxide levels and on lung function. The results were compared with placebo challenge. A total of 28 subjects were divided into three study groups: group 1 had been exposed to occupational mould; group 2 consisted of atopic subjects; and group 3 was a control group. Some 3 h after A. fumigatus challenge, there was a considerable increase in FeNO, and a significant difference was observed between the A. fumigatus and placebo inhalations. The difference was seen in all study groups. No such differences were found in the levels of FnNO or nitrite in nasal lavage fluid. Subjects reported significantly more frequent respiratory tract symptoms after the A. fumigatus inhalation compared with placebo challenge. In conclusion, it was shown here that inhalation challenge of Aspergillus fumigatus elevated fractional exhaled nitric oxide levels. An increase in fractional exhaled nitric oxide may serve as an indicator of respiratory inflammation of acute mould exposure.

Adult↗
PubMed2001

Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation.

Organotypic cocultures of keratinocytes and fibroblasts generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The use of mouse fibroblasts and human keratinocytes facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and growth regulation. Moreover, the functional significance for the keratinocyte phenotype of genetically modified fibroblasts from transgenic or knockout mice, even those exhibiting an embryonic lethal phenotype, can be studied in such heterologous in vitro tissue equivalents. Here we communicate results of such studies revealing the antagonistic function of mouse fibroblasts defective in the AP-1 constituents c-Jun and JunB, respectively, on human keratinocyte growth and differentiation. Furthermore, the hematopoietic growth factor granulocyte macrophage-colony stimulating factor has been identified as a novel regulator of keratinocyte growth and differentiation. As will be reported in detail elsewhere both granulocyte macrophage-colony stimulating factor and keratinocyte growth factor have been identified as major mediators of fibroblast-keratinocyte interactions and their expression is induced via AP-1 by interleukin-1 released by the epithelial cells. Thus, these heterologous cocultures provide a novel promising tool for elucidating molecular mechanisms of epithelial-mesenchymal interactions and their consequences on epithelial cell proliferation and differentiation.

Adult↗
PubMed2000

Keratinocyte growth regulation in defined organotypic cultures through IL-1-induced keratinocyte growth factor expression in resting fibroblasts.

Balanced keratinocyte proliferation and differentiation resulting in regular tissue organization strictly depend on dermal support. Organotypic cultures represent biologically relevant in vitro models to study the molecular mechanism of the underlying dermal-epidermal interactions. To mimic the state of resting fibroblasts in the dermis, postmitotic (irradiated) fibroblasts were incorporated in the collagen matrix, where they typically support epidermal proliferation and tissue organization. In coculture with keratinocytes, fibroblasts exhibit an enhanced expression of keratinocyte growth factor and the interleukin-1 receptor (type I), which further increase with culture time. In cocultured keratinocytes, keratinocyte growth factor receptor as well as RNA expression and protein release of interleukin-1alpha and interleukin-1beta are upregulated. We hypothesized that the modulated cytokine expression represents a basic mechanism for keratinocyte growth regulation. The functional significance of this double paracrine pathway, i.e., induction of keratinocyte growth factor expression in fibroblasts by keratinocytes via release of interleukin-1, was confirmed by interfering with both signaling elements: (i) interleukin-1-neutralizing antibodies and interleukin-1 receptor antagonist significantly inhibited keratinocyte growth factor release, keratinocyte proliferation, and tissue formation comparable to the effect produced by keratinocyte-growth-factor-blocking antibodies; (ii) addition of keratinocyte growth factor to cocultures with inactivated interleukin-1 pathway completely reverted growth inhibition; (iii) in organotypic cocultures with subthreshold fibroblast numbers both interleukin-1 and keratinocyte growth factor restored the impaired epidermal morphogenesis. Thus, epidermal tissue regeneration in organotypic cocultures is mainly regulated by keratinocyte-derived interleukin-1 signaling, which induces keratinocyte growth factor expression in cocultured fibroblasts. This demonstrates a novel role for interleukin-1 in skin homeostasis substantiating data from wound healing studies in vivo.

Cell Differentiation↗
PubMed1999

Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation.

Skin equivalents formed by keratinocytes cocultured with fibroblasts embedded in collagen lattices represent promising tools for mechanistic studies of skin physiology, for pharmacotoxicologic testing, and for the use as skin substitutes in wound treatment. Such cultures would be superior in defined media to avoid interference with components of serum or tissue extracts. Here we demonstrate that a defined medium (supplemented keratinocyte defined medium) supports epidermal morphogenesis in organotypic cocultures equally well as serum-containing medium (mixture of Ham's F12 and Dulbecco's modified Eagle's medium), as documented by hallmarks of the epidermal phenotype studied by immunofluorescence and electron microscopy. In both cases regularly structured, orthokeratinized epithelia evolved with similar kinetics. Morphology in mixture of Ham's F12 and Dulbecco's modified Eagle's medium was slightly hyperplastic, and keratins 1 and 10 synthesis less co-ordinated than in supplemented keratinocyte defined medium, but a consistently inverted sequence of expression of keratins 1 and 10 was found in either medium. The late differentiation markers filaggrin, involucrin, keratin 2e, and transglutaminase 1 corresponded in their typical distribution in upper suprabasal layers. Keratin 16 persisted under both conditions indicating the activated epidermal state. Keratinocyte proliferation was comparable in both media, whereas fibroblast multiplication and proliferation was delayed and reduced in supplemented keratinocyte defined medium. In both media, ultrastructural features of epidermal differentiation as well as reconstitution of a basement membrane occurred similarly. Immature lamellar bodies and cytoplasmatic vacuoles, however, indicated an impaired lipid metabolism in supplemented keratinocyte defined medium. Nevertheless, these defined organotypic cocultures provide a suitable basis for in vitro skin models to study molecular mechanisms of tissue homeostasis and for use in pharmacotoxicologic testing.

Animals↗
PubMed1998

Dynamics of basement membrane formation by keratinocyte-fibroblast interactions in organotypic skin culture.

The cutaneous basement membrane zone, composed of numerous macromolecules, plays a multifunctional role in tissue regeneration and maintenance. To elucidate the cellular origin and dynamics of basement membrane formation, de novo synthesis, deposition, and ultrastructural assembly of its components were analyzed in organotypic cultures of adult skin keratinocytes on collagen gels with or without collagen-embedded dermal cells. Collagen IV and laminin-1 deposition occurred only in the presence of mesenchymal cells: patchy at day 4 and continuous after 1 week. Chain-specific mRNA expression started at day 2 in both keratinocytes and fibroblasts. It steadily increased up to day 10, however, with a reciprocal induction pattern, mRNA abundance shifting from keratinocytes to fibroblasts. On the other hand, laminin-5 staining was first observed at day 4, but in keratinocyte both mono- and cocultures. This was followed by nidogen, which was detected in cocultures but also in dermal monocultures. Laminin-5 protein persisted throughout day 21, whereas nidogen steadily increased in intensity. Expression kinetics revealed high levels of laminin-5 transcripts early and in keratinocytes only, whereas nidogen was expressed later and predominantly in fibroblasts. Although basement membrane protein deposition was continuous at day 14, the ultrastructural organization was still fragmentary, eventually normalizing at 3 weeks. These data demonstrate a dynamic interaction and cooperation of epithelial and mesenchymal skin cells in basement membrane formation. This interaction is supposedly mediated via diffusible factors. Our findings further extend the scope of epithelial-mesenchymal interactions stressing that both cell compartments are essential to constitute a tissue-specific extracellular matrix structure.

Basement Membrane↗
PubMed1998

5-Lipoxygenase expression in Langerhans cells of normal human epidermis.

We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer, in outer hair root sheaths, and in the epithelial compartment of sebaceous glands. Examination of skin specimens by immunohistochemistry and of primary ECs by flow cytometry mapped the 5-LO protein exclusively to Langerhans cells (LCs). The LC 5-LO protein was largely found in the nuclear matrix, in nuclear envelopes, and perinuclear regions as indicated by in situ confocal laser scan microscopy. Reverse transcription-PCR and immunoblot analyses of purified primary EC populations further indicated that LCs are major 5-LO expressing cells. Enriched primary LCs were also found to contain 5-LO activating protein (FLAP), leukotriene (LT) C4 synthase, and LTA4 hydrolase. By contrast, 5-LO, FLAP, and LTC4 synthase were undetectable or largely reduced, but LTA4 hydrolase transcripts and protein were identified in ECs depleted of LCs. These data show that naive LCs are major, and possibly the sole, 5-LO pathway expressing cells in the normal human epidermis.

Arachidonate 5-Lipoxygenase↗
PubMed1998

Epidermal differentiation and basement membrane formation by HaCaT cells in surface transplants.

The immortal human keratinocyte line HaCaT has been employed in many studies as paradigm for epidermal keratinocytes. In order to demonstrate its potential to form stable epidermal structures in response to connective tissue, this was challenged in surface transplants on nude mice, where normal keratinocytes rebuild a typical epidermis within two weeks. During the initial regeneration phase (day 1-4) multilayered but poorly organized epithelia formed with proliferating cells in all layers in analogy to normal keratinocytes. Similarly, with tissue consolidation (around day 7) proliferation was reduced and restricted to cells in basal position marked by keratin K14 and beta1-integrin immunostaining. The strong suprabasal reaction for K1 and K10, the appearance of the late markers K2e, filaggrin and loricrin as well as the polarized distribution of alpha2beta1 and alpha3beta1 indicated advancing tissue normalization (day 14). Keratinization further improved at around three weeks switching from the initial parakeratotic to the regular orthokeratotic type which was prominent at six weeks. Accordingly, most ultrastructural features typical for epidermis or normal keratinocyte grafts were detectable including a complete basement membrane (BM) with regular attachment structures. Matrix- and BM-components appeared sequentially with marked linear deposition of laminin-5 (day 4) followed by accumulation of collagen-IV and 'classical' BM-laminin between one and two weeks. With the general codistribution of integrin alpha6beta4 and BM-molecules (day 14) collagen-VII lining of BM became prominent, while epithelium and host connective tissue were still separated by the collagen matrix. In accordance with the delayed orthokeratinization, wound-matrix molecules (fibronectin, tenascin) persisted longer than in normal keratinocyte transplants. Finally, grafts of long-term passaged (no. 310) cells demonstrated a remarkable stability in the expression of epidermal markers. Thus, the immortalized HaCaT cells reveal a generally high competence to realize an epidermal phenotype in a natural environment and appear therefore qualified for in vitro studies on structural and regulatory aspects of keratinocyte physiology and pathology.

Animals↗
PubMed1998

Plasminogen activator inhibitor type 2 is expressed in keratinocytes during re-epithelialization of epidermal defects.

Plasminogen activation is observed in the human epidermis during re-epithelialization of epidermal defects. The activation reaction depends on plasminogen activators (PAs) associated with re-epithelializing keratinocytes. PA inhibitor type 2 (PAI-2) is thought to be a major epidermal PA inhibitor in keratinocytes. However, no data are available on the expression of PAI-2 in keratinocytes during epidermal regeneration. We have therefore analysed PAI-2 at the mRNA and protein level in keratinocyte cultures as well as in epidermal lesions in which re-epithelializing keratinocytes were apparent. We found that PAI-2 expression at the mRNA and protein level was negatively correlated with the cell density in regular keratinocyte cultures. In organotypic cocultures, in which the transition from a re-epithelializing to a sedentary phenotype can be studied, PAI-2 was most strongly expressed in early cultures prior to formation of a differentiated epidermis-like structure. We found a strong expression of PAI-2 in keratinocytes that re-epithelialized dermal burn wounds or lesions caused by the autoimmune blistering disease pemphigus vulgaris. Our results suggest that not only PAs, but also a major PA inhibitor, PAI-2, are expressed in keratinocytes that are actively involved in re-epithelialization.

Blotting, Northern↗
PubMed1997

Integrin and basement membrane normalization in mouse grafts of human keratinocytes--implications for epidermal homeostasis.

Integrin patterns and formation of basement membrane (BM) were investigated in correlation to epidermal growth and differentiation during skin regeneration in human keratinocyte transplants on nude mice. Immuno-fluorescence and transmission electron microscopy (TEM) showed that different stages of tissue reconstruction were characterized by a sequence of coordinated events. Features of the initial tissue activation, with rapid keratinocyte proliferation around day 4, including cells in a suprabasal position, were: (1) a marked increase in and extended distribution of the integrin chains alpha 2, alpha 3, beta 1 and alpha 6, while beta 4 already showed a preferential basal location; (2) de novo expression of alpha 5 and alpha v; and (3) marked deposition of laminin-5 and nidogen but low levels of other BM components. Tissue normalization during the 2nd week, initiated by a drastic decrease in the number of proliferating cells after day 4, now strictly in basal position, was signified: by (1) orthotopic staining for basal-type keratins (K5, K14) together with a regular pericellular alpha 2 beta 1 and alpha 3 beta 1 distribution, (2) linear, balanced deposition of BM components (e.g. laminin-1, type IV collagen) and (3) colocalization of integrin alpha 6 beta 4 and bullous pemphigoid antigen. Simultaneously at 7 days hemidesmosomes and a defined BM had developed (TEM), becoming continuous at 14 days. This coincided with the regular distribution of suprabasal keratins (K1, K10) as well as intermediate (involucrin) and late differentiation markers (filaggrin, loricrin). Type-VII collagen deposition, still irregular at 14 days, became continuous at 22 days together with developing BM-anchoring fibrils indicating final tissue consolidation. This model mimics principal stages of epidermal wound healing in human skin and implies a linkage between BM assembly, integrin distribution and the compartment of proliferation competent cells, which in turn determines the onset of differentiation. Thus, apart from the balance of diffusible growth regulators, this positional control of keratinocytes, largely accomplished by integrin-matrix interactions, seems to be prerequisite to establishment and maintenance of tissue homeostasis.

Animals↗
PubMed1995

Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.

Arachidonate 5-Lipoxygenase↗
PubMed1995

Differential expression of urokinase-type plasminogen activator (uPA), its receptor (uPA-R), and inhibitor type-2 (PAI-2) during differentiation of keratinocytes in an organotypic coculture system.

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-1 stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.

Animals↗
PubMed1995

Differentiation and tumor progression.

Clinical and experimental experience indicate that differentiation and malignancy are inversely correlated. However, more recent experimental studies using mouse and human keratinocyte systems have demonstrated that complete or even substantial loss in overall epithelial differentiation is not a prerequisite for malignant growth of cancer cells. Major defects in differentiation are also not a prerequisite for premalignant stages, in particular for cell immortalization, which is considered an early and essential step in the transformation process. Moreover, progressive dedifferentiation, often associated with advanced tumor stages, is also found in immortalized cell lines which are, however, nontumorigenic. On the other hand, malignant cell lines may have maintained a high degree of their normal differentiation program and sensitivity to differentiation modulators. However, to date no transformed keratinocyte cell lines with completely normal differentiation have been observed. Since epidermal keratinization is a very complex process involving many different parameters and is fully expressed only under in vivo conditions, an exact and quantitative comparison of such ill-defined phenomena (differentiation and malignancy) is still problematic. Obviously, both phenomena are under separate control and not causally linked. Nevertheless, a better understanding of factors and mechanisms regulating differentiation and of their disturbance in carcinogenesis would offer new possibilities to design novel tumor therapeutic strategies in the field of differentiation therapy.

Biomarkers↗
PubMed1994

Modulation of the differentiated phenotype of keratinocytes of the hair follicle and from epidermis.

The differentiation capacity and its modulation by cell-cell and cell-matrix interactions of epithelial cells from epidermis (NEK), hair follicle outer root sheath (ORS) and hair matrix cells (HMC) in different experimental model systems is reviewed. Reformation of structurally intact and functioning hair follicles has been achieved with isolated neonatal mouse cells in vivo when follicular epithelia and dermal fibroblasts were transplanted. This structural reorganization has not yet been feasible in vitro in either surface or matrix embedded organotypic cocultures with mesenchymal cells. While the epithelial cells isolated from epidermis or hair follicle compartments formed similar stratified and keratinizing epithelia in vitro, their degree of differentiation was significantly different, declining from NEK over ORS to HMC. Differentiation was further reduced in HMC cultures in the presence of dermal papilla cells (DPC). Differentiation was analyzed by morphologic criteria and the biochemical analysis as well as immunohistochemical localization of differentiation products such as keratins, involucrin, filaggrin, integrins, basement membrane components and membrane antigens. The results demonstrate fully maintained differentiation capacity of skin and appendage-keratinocytes to reconstitute a squamous epithelium, reflecting their common origin from embryonic epidermis. For the induction of hair follicle-specific structural and functional characteristics, different and probably more complex interactive mechanisms may be required.

Animals↗
PubMed1993

Differential modulation of epidermal keratinization in immortalized (HaCaT) and tumorigenic human skin keratinocytes (HaCaT-ras) by retinoic acid and extracellular Ca2+.

The growth and differentiation response to retinoic acid (RA) was studied in the human keratinocyte line HaCaT and tumorigenic clones transfected with c-Ha-ras oncogene (HaCaT-ras). Differentiation (mainly keratin synthesis) was evaluated and correlated to cell proliferation in vitro but also growth behaviour in vivo (tumorigenicity). Comparable to normal keratinocytes, HaCaT cells and ras clones showed increased expression of the epidermal suprabasal keratins K1 and K10 upon RA depletion of the media (delipidized serum), while simple epithelial type keratins K7, K8 and K18 as well as K19 and K13 (typical of internal stratified epithelia) were almost completely suppressed. The cell density-dependent increase of K1 and K10 at intermediate RA levels (as in regular media with untreated serum) was also observed at Ca2+ levels below 0.1 mM, thus being clearly unrelated to stratification, whereas K13 synthesis was Ca(2+)-dependent and initiated with stratification. The effects on keratins were fully reversed by increasing RA concentrations. There was only mild stimulation of proliferation at RA doses (10(-10) to 10(-8) M) not directly corresponding to suppression of keratinization. Thus, the negative RA influence on K1 and K10, opposed to the effect on simple keratins, substantiates the preserved regulatory capacity rendering these cells appropriate models for biological testing. Among the various tumorigenic HaCaT-ras clones highly and moderately differentiating ones could be distinguished, accordingly induction in vitro led to a comparable spectrum of differentiation markers (K1 and K10 appearing early, and filaggrin late) as growth in vivo. These in vitro results demonstrate that, in spite of some differences in RA sensitivity, virtually all clones possess the epidermal differentiation repertoir which is regulated according to the same principles. Finally, this confirms our in vivo data that differentiation potential is not inversely related to the state of transformation or tumorigenicity.

Calcium↗
PubMed1991

Experimental modulation of the differentiated phenotype of keratinocytes from epidermis and hair follicle outer root sheath and matrix cells.

Follicles of human anagen hair were separated into morphologically distinct compartments (by sequential trypsinization and microdissection) for the biochemical and immunological analysis of keratins as differentiation markers to diagnose the type of epithelial differentiation. While outer root sheath contained throughout the "soft" (cyto)keratins K5, 6, 14, 16, and 17, and hair cortex contained exclusively a set of acidic and basic "hard" alpha-keratins (consistent up to the hair tip), in inner root sheath and hair cuticle peptides related or derived from suprabasal epidermal keratins K1 and 10 were detected. These keratin profiles served as in vivo correlates for the evaluation of type and degree of differentiation achieved by the respective isolated epithelial cells, comparing different growth or culture conditions. Cultures of ORS cells and hair matrix cells (PHS cells) as well as normal keratinocytes were initiated using postmitotic human dermal fibroblasts as efficient feeder cells. On lifted collagen gels populated with HDF ("surface" cultures), ORS and PHS cells formed stratified epithelial expressing epidermal differentiation markers such as keratins K1 and 10, involucrin, and filaggrin. Compared with NEK "surface" cultures, balance between growth and differentiation was better maintained by both follicular cell types. In contrast, epidermal tissue homeostasis was largely normalized in transplants on nude mice regardless of the epithelial cell type, apparent from orderly tissue structure, regular distribution of keratin K10, filaggrin, and involucrin, and distinct continuous deposition of basement membrane components at the epithelium-collagen interface. Embedded in Matrigel (on top of HDF collagen gels) ORS cells and NEK formed spheroids exhibiting inward-directed epidermoid differentiation, increasing with time. All epidermal maturation products found in "surface" cultures were likewise expressed, and again differentiation greatly outbalanced proliferation in spheroids of NEK but not of ORS cells. PHS cells embedded together with HDF in Matrigel produced similar spheroids as ORS cells. Size of spheroids and degree of epidermoid differentiation were dramatically reduced when HDF were replaced by follicular DP cells, demonstrating the crucial role of the mesenchymal "companion" cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Differentiation↗
PubMed1991

Epidermal morphogenesis and keratin expression in c-Ha-ras-transfected tumorigenic clones of the human HaCaT cell line.

Several tumorigenic (benign and malignant) clones have been raised from the human epidermal cell line HaCaT after transfection with the c-Ha-ras oncogene (val 12) (P. Boukamp et al., Cancer Res., 50: 2840-2847, 1990). In culture, these HaCaT-ras clones expressed epidermal differentiation markers, such as keratins K1 and 10, at high density or upon depletion of retinoic acid. Accordingly, as HaCaT cells, the clones formed well-differentiated stratified epithelia synthesizing K1 and 10 in surface transplants, while simple and internal epithelial keratins seen in culture were suppressed (as upon retinoic acid depletion in vitro). In transplants of HaCaT cells, in contrast to those of normal keratinocytes, K1 appeared prematurely already in basal cells, while K10 localized rather normally in the suprabasal position. Keratins 1 and 10 were also synthesized in transplants of HaCaT-ras clones (again K1 preceding K10), but both generally shifted toward upper layers. This was particularly evident in thicker transplants of malignant clones. Staining for both keratins persisted "suprabasally" in invasive tissue masses, and this corresponded to their marked expression in solid carcinomas (after s.c. injection), seen by immunofluorescence and two-dimensional gel electrophoresis. Thus, notwithstanding some variations, differentiation potential was not significantly reduced in these clones disregarding levels of ras oncogene expression and malignant properties.

Cell Line↗
PubMed1990

Mesenchyme-mediated and endogenous regulation of growth and differentiation of human skin keratinocytes derived from different body sites.

In culture, keratinocytes generally express aberrant growth and differentiation programs, which are largely normalized in cell transplants. In order to study the underlying regulatory phenomena and to distinguish between intrinsic properties and external factors, different in vitro and in vivo models have been applied using human keratinocytes from foreskin and trunk skin. When transplanted onto nude mice, keratinocytes reformed a regular epithelium with expression of the differentiation markers, keratins K1 and K10, involucrin and filaggrin. Tissue homeostasis improved in later transplants, as made apparent by coexpression and regular distribution of K1 and K10. Since this was achieved in transplants, whether in contact with mesenchyme or separated by collagen matrix, renormalization was obviously mediated by diffusible factors. In vitro, the host-mesenchymal influence could largely be mimicked by recombining organotypic cultures (keratinocytes on lifted collagen gels) with de-epidermized dermis, but tissue homeostasis was apparently not achieved. Comparing keratinocytes from trunk skin and foreskin, differences observed in situ persisted in isolated cells and reconstituted tissues. The hyperproliferative character of foreskin epidermis, with its less-pronounced stratum granulosum, was maintained in recombinant cultures and transplants along with the expression of keratin K13 (typical for foreskin in situ) irrespective of the type of mesenchyme. Thus, we could demonstrate with these model systems that: (a) the regulation of keratinocyte growth and differentiation is mesenchyme-dependent; (b) it is mediated by diffusible factors; but that (c) differences between epidermis of different body sites are also controlled by intrinsic programs.

Adolescent↗