Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.     

Role of leukemia inhibitory factor in the regulation of the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts/melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanoblast/melanocyte-proliferation medium supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Leukemia inhibitory factor (LIF) supplemented to the medium from initiation of primary culture increased the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. Pure cultured primary melanoblasts or melanocytes were further cultured with the medium supplemented with LIF from 14 days (keratinocyte depletion). LIF stimulated the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes in the absence of keratinocytes. Moreover, anti-LIF antibody supplemented to the medium from initiation of primary culture inhibited the proliferation of melanoblasts or melanocytes as well as the differentiation of melanocytes. These results suggest that LIF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.     

BALB and kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent BALB/c mouse epidermal keratinocyte lines

Clonal BALB/c mouse epidermal keratinocyte (BALB/MK) cell lines were established in tissue culture. Despite their aneuploid nature, the lines were nontumorigenic, and retained in vitro properties similar to those of primary diploid keratinocytes. These included the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0 mM in the medium. The cells also demonstrated an absolute requirement for nanomolar concentrations of epidermal growth factor (EGF) for their proliferation. BALB or Kirsten murine sarcoma viruses are acute transforming retroviruses, which have been shown to transform fibroblastic and hematopoietic cells. Infection of BALB/MK or its clonal sublines with either virus leads to the rapid acquisition of EGF-independent growth. The cells concomitantly lose their sensitivity to calcium-induced terminal differentiation. Thus these retroviruses can rapidly confer upon epithelial keratinocytes in culture growth properties that resemble those of malignant epidermoid carcinoma cells.     

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

Hepatocyte growth factor controls the proliferation of cultured epidermal melanoblasts and melanocytes from newborn mice

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with hepatocyte growth factor (HGF) from 14 days (keratinocyte depletion). The HGF increased the number of melanoblasts and melanocytes, but not the percentage of differentiated melanocytes in the melanoblast-melanocyte population in the absence of keratinocytes. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and/or G2/M phases of the cell cycle were increased by the treatment with HGF. Moreover, an anti-HGF antibody supplemented to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes, but not the differentiation of melanocytes. These results suggest that HGF is a keratinocyte-derived factor involved in regulating the proliferation of epidermal melanoblasts and melanocytes from newborn mice in cooperation with cAMP elevators and/or bFGF.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

An unusual strain of human keratinocytes which do not stratify or undergo terminal differentiation in culture

We have characterized an unusual cell phenotype in third passage cultures of a human keratinocyte strain derived from newborn foreskin epidermis. The cells had the same DNA fingerprint pattern as the second passage, morphologically normal, keratinocytes; they formed desmosomes and expressed the keratin profile characteristic of normal keratinocytes in culture. However, unlike normal keratinocytes, the cells did not grow as compact colonies and did not stratify or undergo terminal differentiation, even after TPA treatment or suspension culture. For these reasons we named them ndk for "nondifferentiating keratinocytes." The ndk cells also differed from normal keratinocytes in that they did not require a feeder layer and were not stimulated by cholera toxin to proliferate. The ndk cells had an absolute requirement for hydrocortisone and their growth rate was increased when epidermal growth factor was added to the medium. Although ndk failed to undergo terminal differentiation in culture, they were not transformed, since they were still sensitive to contact inhibition of growth, did not proliferate in soft agar, and had a limited lifespan in culture. The appearance of the ndk phenotype was correlated with a doubling of chromosome number and the presence of a lp marker chromosome. We suggest that these cells are a useful experimental adjunct to cultures of normal keratinocytes, in which proliferation and terminal differentiation are tightly coordinated, because in ndk cells there appears to be a block in terminal differentiation.     

Effects of low frequency pulsed electrical current on keratinocytes in vitro

The effects of low frequency pulsed electrical current on epidermal repair in vitro were examined. Charge-balanced current stimuli proposed for chronic wound treatment were tested on skin keratinocytes cultured at an air-liquid interface on dead human dermis. Results imply that the balance between proliferation and differentiation in electrically treated samples is significantly modified in favor of differentiation. More advanced differentiation, shown through epidermal histology, was obtained in cultures exposed to electrical current, whereas the culture growth, the result of keratinocyte migration and proliferation, was greater in control samples. Bioelectromagnetics 18:250–254, 1997. © 1997 Wiley-Liss, Inc.     

Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.     

Site-matched papillary and reticular human dermal fibroblasts differ in their release of specific growth factors/cytokines and in their interaction with keratinocytes

The interfollicular dermis of adult human skin is partitioned into histologically and physiologically distinct papillary and reticular zones. Each of these zones contains a unique population of fibroblasts that differ in respect to their proliferation kinetics, rates at which they contract type I collagen gels, and in their relative production of decorin and versican. Here, site-matched papillary and reticular dermal fibroblasts couples were compared to determine whether each population interacted with keratinocytes in an equivalent or different manner. Papillary and reticular fibroblasts grown in monolayer culture differed significantly from each other in their release of keratinocyte growth factor (KGF) and granulocyte-macrophage colony stimulating factor (GM-CSF) into culture medium. Some matched fibroblast couples also differed in their constitutive release of interleukin-6 (IL-6). Papillary fibroblasts produced a higher ratio of GM-CSF to KGF than did corresponding reticular fibroblasts. Interactions between site-matched papillary and reticular couples were also assayed in a three-dimensional culture system where fibroblasts and keratinocytes were randomly mixed, incorporated into type I collagen gels, and allowed to sort. Keratinocytes formed distinctive cellular masses in which the keratinocytes were organized such that the exterior most layer of cells exhibited characteristics of basal keratinocytes and the interior most cells exhibited characteristics of terminally differentiated keratinocytes. In the presence of papillary dermal fibroblasts, keratinocyte masses were highly symmetrical and cells expressed all levels of differentiation markers. In contrast, keratinocyte masses that formed in the presence of reticular fibroblasts tended to have irregular shapes, and terminal differentiation was suppressed. Furthermore, basement membrane formation was retarded in the presence of reticular cells. These studies indicate that site-matched papillary and reticular dermal fibroblasts qualitatively differ in their support of epidermal cells, with papillary cells interacting more effectively than corresponding reticular cells.     

Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and a melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with granulocyte-macrophage colony-stimulating factor (GMCSF) from 14 days (keratinocyte depletion). GMCSF stimulated the number of melanoblasts/melanocytes as well as the percentage of differentiated melanocytes in keratinocyte-depleted cultures. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and G(2)/M phases of the cell cycle were increased by the treatment with GMCSF. Moreover, anti-GMCSF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts/melanocytes as well as the differentiation of melanocytes. Enzyme-linked immunosorbent assay of culture media revealed that GMCSF was secreted from keratinocytes, but not from melanocytes. These results suggest that GMCSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanoblasts/melanocytes in culture in cooperation with cAMP elevator and bFGF.     

Mesenchymal stem cells differentiate into keratinocytes and express epidermal kallikreins: Towards an in vitro model of human epidermis

Epidermal differentiation is a complex process in which keratinocytes go through morphological and biochemical changes in approximately 15 to 30 days. Abnormal keratinocyte differentiation is involved in the pathophysiology of several skin diseases. In this scenario, mesenchymal stem cells (MSCs) emerge as a promising approach to study skin biology in both normal and pathological conditions. Herein, we have studied the differentiation of MSC from umbilical cord into keratinocytes. MSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (proliferation medium) and, after characterization, differentiation was induced by culturing cells in a defined keratinocyte serum-free medium (KSFM) supplemented with epidermal growth factor (EGF) and calcium chloride ions. Cells cultivated in DMEM were used as control. Cultures were evaluated from day 1 to 23, based on the cell morphology, the expression of p63, involucrin and cytokeratins (KRTs) KRT5, KRT10 and KRT14, by quantitative polymerase chain reaction, Western blot analysis or immunofluorescence, and by the detection of epidermal kallikreins activity. In cells grown in keratinocyte serum-free medium with EGF and 1.8 mM calcium, KRT5 and KRT14 expression was shown at the first day, followed by the expression of p63 at the seventh day. KRT10 expression was detected from day seventh while involucrin was observed after this period. Data showed higher kallikrein (KLK) activity in KSFM-cultured cells from day 11th in comparison to control. These data indicate that MSC differentiated into keratinocytes similarly to that occurs in the human epidermis. KLK activity detection appears to be a good methodology for the monitoring the differentiation of MSC into the keratinocyte lineage, providing useful tools for the better understanding of the skin biology.     

Specific changes of Ras GTPase-activating protein (GAP) and a GAP-associated p62 protein during calcium-induced keratinocyte differentiation.

Induction of tyrosine phosphorylation occurs as an early and specific event in keratinocyte differentiation. A set of tyrosine-phosphorylated substrates which transduce mitogenic signals by tyrosine kinases has previously been identified. We show here that of these substrates, the Ras GTPase-activating protein, GAP, is specifically affected during calcium-induced keratinocyte differentiation. As early as 10 min after calcium addition to cultured primary mouse keratinocytes, GAP associates with tyrosine-phosphorylated proteins and translocates to the membrane. In addition, a GAP-associated protein of approximately 62 kDa (p62) becomes rapidly and heavily tyrosine phosphorylated in both membrane and cytosolic fractions. This protein corresponds to the major tyrosine-phosphorylated protein that is induced in differentiating keratinocytes as early as 5 min after calcium addition. p62 phosphorylation was not observed after exposure of these cells to epidermal growth factor, phorbol ester, or transforming growth factor beta. In contrast, PLC gamma and P13K were tyrosine phosphorylated after epidermal growth factor, but not calcium, stimulation. Thus, changes of Ras GAP and an associated p62 protein occur as early and specific events in keratinocyte differentiation and appear to involve a calcium-induced tyrosine kinase.     

钙离子对鼠角质细胞生长和分化的影响

用无血清培养基培养角质细胞,研究了Ca²⁺对鼠角质细胞生长和分化的影响。实验结果表明,培养基中钙离子最佳浓度为0.2mmol/L。在此浓度下,细胞克隆形成率达到10.8%,细胞的贴壁率达到28.7%,细胞的分化比例和老化比例分别为5.4%和26.3%;当Ca²⁺浓度达到0.6mmol/L以上时,则会引起角质细胞显著的分化和老化。     

Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16(INK4a) cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16(INK4a)-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT(+) keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16(INK4a) expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16(INK4a)-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.     

Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11(th) to 15(th) day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage.     

Members of the src and ras oncogene families supplant the epidermal growth factor requirement of BALB/MK-2 keratinocytes and induce distinct alterations in their terminal differentiation program.

BALB-/MK-2 mouse epidermal keratinocytes required epidermal growth factor for proliferation and terminally differentiated in response to high Ca2+ concentration. Infection with retroviruses containing transforming genes of the src and ras oncogene families led to rapid loss of epidermal growth factor dependence, in some cases, accompanied by alterations in cellular morphology. The virus-altered cells continued to proliferate in the presence of high levels of extracellular calcium but exhibited alterations in normal keratinocyte terminal differentiation that appear to be specific to the particular oncogene. These alterations bore similarities to abnormalities in differentiation observed in naturally occurring squamous epithelial malignancies.     

Epidermal morphogenesis in an <Emphasis Type="BoldItalic">in-vitro</Emphasis> model using a fibroblasts-embedded collagen scaffold

Summary A novel culture system included a self-designed bi-layer 3-D collagen scaffold with different pore size on both sides and specific culture media for different culture stages. This skin equivalent culture model provides a new investigating system to study the role of extracellular matrix and growth factors including epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor beta 1 (TGF-β1), in the cell–cell and cell–matrix interactions. Keratinocytes were seeded onto the dermal equivalent and incubated under submerged condition for 5 days then proceeding to air–liquid interface cultured either with or without EGF addition. In this study, EGF has a positive effect on the keratinocyte migration and proliferation in the submerged stage. However, when 10 ng per ml of EGF was continual added in the air-lifted stage, a less organized and thin differentiated keratinocyte layers were found. Continual 10 ng per ml of EGF addition in the air-lifted stage resulted in uneven cell–matrix interface, and disorganization of the suprabasal layers. On the contrary, in the air-lifted stage without excess EGF, the epithelium cells will stratify, differentiate, and form an epidermis completed with basal, spinous, granular, and cornified layers. The results showed that time scale modulation of EGF on keratinocyte cell behavior depend on the expression of paracrine or autocrine growth factors (e.g. KGF and TGF-β1).     

Transforming growth factor-beta-activated kinase 1 is essential for differentiation and the prevention of apoptosis in epidermis

Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the mitogen-activated protein (MAP) kinase family and is an upstream signaling molecule of nuclear factor-kappaB (NF-kappaB). Given that NF-kappaB regulates keratinocyte differentiation and apoptosis, TAK1 may be essential for epidermal functions. To test this, we generated keratinocyte-specific TAK1-deficient mice from Map3k7(flox/flox) mice and K5-Cre mice. The keratinocyte-specific TAK1-deficient mice were macroscopically indistinguishable from their littermates until postnatal day 2 or 3, when the skin started to roughen and wrinkle. This phenotype progressed, and the mice died by postnatal day 7. Histological analysis showed thickening of the epidermis with foci of keratinocyte apoptosis and intra-epidermal micro-abscesses. Immunohistochemical analysis showed that the suprabasal keratinocytes of the TAK1-deficient epidermis expressed keratin 5 and keratin 14, which are normally confined to the basal layer. The expression of keratin 1, keratin 10, and loricrin, which are markers for the suprabasal and late phase differentiation of the epidermis, was absent from the TAK1-deficient epidermis. Furthermore, the TAK1-deficient epidermis expressed keratin 16 and had an increased number of Ki67-positive cells. These data indicate that TAK1 deficiency in keratinocytes results in abnormal differentiation, increased proliferation, and apoptosis in the epidermis. However, the keratinocytes from the TAK1-deficient epidermis induced keratin 1 in suspension culture, indicating that the TAK1-deficient keratinocytes retain the ability to differentiate. Moreover, the removal of TAK1 from cultured keratinocytes of Map3k7(flox/flox) mice resulted in apoptosis, indicating that TAK1 is essential for preventing apoptosis. In conclusion, TAK1 is essential in the regulation of keratinocyte growth, differentiation, and apoptosis.     

Cathepsin B is essential for regeneration of scratch-wounded normal human epidermal keratinocytes

Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.