Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.     

Isolation and propagation of keratinocytes derived from Cashmere goat fetus

The study was conducted to isolate epidermal keratinocytes from Cashmere goat fetus with the aim to develop suitable conditions for keratinocyte cultivation and propagation. The methods developed for keratinocyte culture include (i) use of a feeder-layer of mitotically inactivated fibroblasts obtained from goat and mouse fetal skin, (ii) use of a substrate such as collagen IV, or (iii) without use of any substrate. Epidermal cell removal was established by enzymatically separating keratinocytes from 12 to 16 weeks aged fetal skin tissues treated with 0.125% trypsin solution overnight at 4 degrees C. The cells were maintained in all culture conditions with serum containing medium. Keratinocyte multiplication and proliferation were comparable in different culture conditions and the improved cellular attachment and growth have been obtained in cultures on feeder layers. Colony forming keratinocytes on feeder layer were heterogeneous in their growth potential. In feeder free conditions, high cellular density was required at plating for sub-cultivation as their poor attachment in culture dishes. This study reports the comparative efficacy of different culture conditions for keratinocyte isolation and in vitro propagation originating from Cashmere goat fetus.     

Effects of feeder layers made of human,mouse, hamster,and rat cells on the cloning efficiency of transformed human cells

Summary The effect of feeder layers on cloning efficiency of transformed human cells was investigated. Embryonic human skin or lung fibroblasts; adult human skin fibroblasts; early passage cells from embryos of mouse, rat, and hamster; established mouse cell lines; 3T3 and 10T1/2 were used as feeder layers after they were lethally exposed to Co-60 gamma-rays at 3,000 rad. As test cells to study the effect of feeder layers on cloning efficiency, WI-38 CT-1 cells transformed in vitro by Co-60 gamma-rays and HGC cells cultured from a human gastric cancer were used. The effect of feeder layers on the cloning efficiency of the test cells was dependent on cell density of feeder layer cells, sources of the feeder layer cells, and kinds of test cells. An optimal density of feeder cels produced cloning efficiencies 3 to 15 times higher than in cultures without a feeder layer. Generally, high density of cells in feeder layers decreased the cloning efficiency of the test cells, presumably owing to contact inhibition of growth and depletion of essential nutrients by the feeder layer cells. Regarding the effect of the feeder layers made of human fibroblasts, there were no significant differences in population doubling levels; tissue origins of fibroblasts; or fibroblasts derived from normal individuals, patients with cancer, or with a genetically high familial incidence of cancer, hereditary adenomatosis of the colon and rectum. This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan.     

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.     

Hyperproliferation of normally quiescent keratinocytes in non-lesional psoriatic skin due to high calcium concentration (an organotypic culture model)

Calcium plays an important role in the regulation of different functions of keratinocytes. In the present work we studied the effect of different extracellular calcium concentrations (0.01 mM-2.0 mM) on the proliferation and differentiation of human keratinocytes in normal human and non-lesional psoriatic skin. Using explant culture model, the proliferative and differentiated subsets of keratinocytes were detected by specific antibodies related to cell proliferation [beta-1 integrin (CD29), proliferating cell antigen (Ki67), proliferating cell nuclear antigen (PCNA)] and differentiation [differentiated cell cytokeratins (K1/K10) and differentiating cell antigen (lectin Ulex europaius agglutinin, UEA-1)]. After 4 days of culturing at high Ca2+ (2.0 mM) we observed marked hyperproliferation among the normally quiescent keratinocytes of non-lesional psoriatic skin. In normal uncultured and cultured skin and in uncultured and two-day-cultured non-lesional psoriatic skin both at normal (1.2 mM) and at high (2.0 mM) Ca2+ concentration only one layer of basal CD29+/Ki67+/K1/K10-/UEA-1- cell was observed. In sections from non-lesional psoriatic skin cultured for 4 days in the presence of high Ca2+ (2.0 mM) this cell population has expanded from at least three layers above the basement membrane. This expanded cell population of the 4-day high Ca2+ cultured non-lesional skin showed clear PCNA positive staining on frozen sections with the strongest positivity among the most basal localized cells. These data suggest that (i) extracellular Ca2+ concentration can influence the proliferation of basal ("stem") keratinocytes, (ii) the proliferative response to high Ca2+ concentration of psoriatic non-lesional basal keratinocytes differs from that of normal basal keratinocytes, (iv) changes in the extracellular Ca2+ milieu might play a role in the induction of the hyperproliferative psoriatic lesion.     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

NG2 proteoglycan expression in mouse skin: altered postnatal skin development in the NG2 null mouse.

In early postnatal mouse skin, the NG2 proteoglycan is expressed in the subcutis, the dermis, the outer root sheath of hair follicles, and the basal keratinocyte layer of the epidermis. With further development, NG2 is most prominently expressed by stem cells in the hair follicle bulge region, as also observed in adult human skin. During telogen and anagen phases of the adult hair cycle, NG2 is also found in stem cell populations that reside in dermal papillae and the outer root sheaths of hair follicles. Ablation of NG2 produces alterations in both the epidermis and subcutis layers of neonatal skin. Compared with wild type, the NG2 null epidermis does not achieve its full thickness due to reduced proliferation of basal keratinocytes that serve as the stem cell population in this layer. Thickening of the subcutis is also delayed in NG2 null skin due to deficiencies in the adipocyte population.     

Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum.     

c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny

BACKGROUND: The epidermis is maintained throughout adult life by pluripotential stem cells that give rise, via daughter cells of restricted self-renewal capacity and high differentiation probability (transit-amplifying cells), to interfollicular epidermis, hair follicles, and sebaceous glands. In vivo, transit-amplifying cells are actively cycling, whereas stem cells divide infrequently. Experiments with cultured human keratinocytes suggest that c-Myc promotes epidermal-stem cell differentiation. However, Myc is a potent oncogene that suppresses differentiation and causes reversible neoplasia when expressed in the differentiating epidermal layers of transgenic mice. To investigate the effects of c-Myc on the stem cell compartment in vivo, we targetted c-MycER to the basal layer of transgenic mouse epidermis. RESULTS: The activation of c-Myc by the application of 4-hydroxy-tamoxifen caused progressive and irreversible changes in adult epidermis. Proliferation was stimulated, but interfollicular keratinocytes still underwent normal terminal differentiation. Hair follicles were abnormal, and sebaceous differentiation was stimulated at the expense of hair differentiation. The activation of c-Myc by a single application of 4-hydroxy-tamoxifen was as effective as continuous treatment in stimulating proliferation and sebocyte differentiation, and the c-Myc-induced phenotype continued to develop even after the grafting of treated skin to an untreated recipient. CONCLUSIONS: We propose that transient activation of c-Myc drives keratinocytes from the stem to the transit-amplifying compartment and thereby stimulates proliferation and differentiation along the epidermal and sebaceous lineages. The ability, demonstrated here for the first time, to manipulate exit from the stem cell compartment in vivo will facilitate further investigations of the relationship between stem cells and cancer.     

A co-cultured skin model based on cell support membranes

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals.     

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.     

Depletion of 43-kD growth-associated protein in primary sensory neurons leads to diminished formation and spreading of growth cones

Epithelial-mesenchymal interactions control epidermal growth and differentiation, but little is known about the mechanisms of this interaction. We have examined the effects of human dermal microvascular endothelial cells (DMEC) and fibroblasts on keratinocytes in conventional (feeder layer) and organotypic cocultures (lifted collagen gels) and demonstrated the induction of paracrine growth factor gene expression. Clonal keratinocyte growth was similarly stimulated in cocultures with irradiated DMEC and fibroblasts as feeder cells. This effect is most probably caused by induction of growth factor expression in cocultured dermal cells. Keratinocytes stimulated mRNA levels for KGF and IL-6 in both mesenchymal cell types and GM-CSF in fibroblasts. The feeder effect could not be replaced by conditioned media or addition of isolated growth factors. In organotypic cocultures with keratinocytes growing on collagen gels (repopulated with dermal cells), a virtually normal epidermis was formed within 7 to 10 d. Keratinocyte proliferation was drastically stimulated by dermal cells (histone 3 mRNA expression and BrdU labeling) which continued to proliferate as well in the gel. Expression of all typical differentiation markers was provoked in the reconstituted epithelium, though with different localization as compared to normal epidermis. Keratins K1 and K10 appeared coexpressed but delayed, reflecting conditions in epidermal hyperplasia. Keratin localization and proliferation were normalized under in vivo conditions, i.e., in surface transplants on nude mice. From these data it is concluded that epidermal homeostasis is in part controlled by complex reciprocally induced paracrine acting factors in concert with cell-cell interactions and extracellular matrix influences.     

Freshly isolated, murine neonatal T cells produce IL-4 in response to anti-CD3 stimulation.

In previous studies of chimeric animals, we found that fetal intrathymic T cell precursors give rise to phenotypically abnormal peripheral T cell populations. Because most peripheral T lymphocytes in newborn mice are the progeny of fetal T cell precursors, this result led to the hypothesis that neonatal and adult T cells differ in their functional capacities. To investigate this issue, the responses of neonatal and adult T cells to anti-CD3 antibody and TCR-independent stimulation were compared. When stimulated with soluble anti-CD3 antibody in the presence of adult accessory cells, neonatal T cell proliferation was markedly decreased compared with that of adult T cells. This reduction in proliferation was associated with both quantitative and qualitative differences in lymphokine production. At 48 h of stimulation with anti-CD3 antibody, neonatal T cells produced at least 10-fold less IL-2 than adult T cells. This apparently accounted for their reduced proliferation because the addition of exogenous IL-2 restored their proliferation to the levels achieved by adult T cells. In striking contrast to adult T cells, neonatal T cells secreted large amounts of IL-4 upon primary stimulation in vitro. The differences between neonatal and adult T cells in proliferation and lymphokine production were shown to be specific for CD3-mediated stimulation. In the presence of phorbol ester and calcium ionophore, neonatal and adult T cells showed equivalent proliferation and IL-2 production. Under these conditions, IL-4 production by neonatal or adult T cells was essentially undetectable. Thus, in response to TCR-independent stimulation, freshly isolated neonatal and adult T cells show similar functional responses. However, when stimulation occurs via the CD3 components of the TCR, the responses of neonatal T cells resemble those of primed T cells from adult animals.     

Differentiation induction of human keratinocytes by phosphatidylethanolamine-binding protein

Phosphatidylethanolamine-binding protein (PEBP) has been demonstrated to bind to Raf-1 and mitogen-activated protein kinase kinase, components of the extracellular signal-regulated protein kinase (ERK) pathway, thereby inhibiting the pathway and resulting in the suppression of cell proliferation. In the present study, we examined whether PEBP is involved in differentiation induction of human keratinocytes. PEBP expression was immunohistochemically examined in normal human skin and skin cancers with different differentiation properties. PEBP was not expressed in the basal layer of the epidermis but was expressed in the spinous and granular layers of normal skin. The protein was expressed in differentiated but not in undifferentiated carcinoma. PEBP expression was also examined in cultured normal human epidermal keratinocytes in which differentiation was induced by calcium treatment. Involucrin was used as a differentiation marker for spinous and granular cells. Northern blotting analysis indicated that both PEBP and involucrin mRNAs were enhanced 6 h after treatment with 2.0 mM CaCl(2). The protein amount of PEBP was also increased by this treatment. To investigate whether PEBP is involved in differentiation induction of keratinocytes, HaCaT keratinocytes were transfected with an expression vector. Fluorescent immunostain revealed that cells expressing PEBP exhibited enlarged and flattened cell shape, and induction of involucrin expression was demonstrated by immunoblot analysis. Although the protein amount of ERK was not altered, phosphorylated ERK levels were decreased and cell proliferation was partly inhibited by PEBP expression. These results indicate that PEBP not only inhibits cell proliferation but also induces differentiation of human keratinocytes.     

p63 expression during normal cutaneous wound healing in humans

p63, a recently identified member of the p53 family, was shown to play a role in morphogenesis and, probably, in tumors of keratinocyte origin. Because p63 seems to be a marker of keratinocytes with a high proliferative potential, the expression of this protein was studied along with another marker of cell proliferation, Ki67, during normal epidermal regeneration in humans. Serial biopsies of human skin healing by a secondary intention were taken at various time intervals (between days 2 and 21 after the injury) and were studied immunohistochemically with the use of a 4A4 monoclonal antibody against the DeltaNp63 variant and MM1 monoclonal antibody against the Ki67 antigen. In the normal and injured skin, the expression of the DeltaNp63 protein was restricted to the epidermal keratinocytes and hair follicle keratinocytes. In the first days of the healing process, there was a dramatic down-regulation of both DeltaNp63 and Ki67 expression in the area of the epidermal tongue invading under the crust. Five days after the injury, induction of DeltaNp63 in the basal keratinocytes could be detected, followed by a gradual increase of its expression in subsequent days. Several days after complete wound closure, DeltaNp63 was still strongly expressed not only in the basal keratinocytes but also in the entire spinous layer, whereas the Ki67 expression was restricted to single cells in the basal layer. The results indicate that DeltaNp63 could be involved in the control of physiologic processes, such as cell proliferation and migration, related to epidermal repair during healing of normal skin in humans.     

A new technique for primary hepatocyte expansion in vitro

The current application for many potential cell-based treatments for liver failure is limited by the low availability of mature functional hepatocytes. Although adult hepatocytes have a remarkable ability to proliferate in vivo, attempts to proliferate adult hepatocytes in vitro have been less successful. In this study, we investigated the effect of coculture cell type on the proliferative response and the functional activities of hepatocytes. We show, for the first time, a robust proliferative response of primary adult rat hepatocytes when cocultured with mouse 3T3-J2 fibroblasts. Hepatocytes cultured at low density on growth-arrested 3T3-J2 fibroblast feeder layers underwent significantly higher proliferation rates than when cultured on feeder layers made of four other cell types. Increasing colony size correlated with an increase in hepatocellular functions. The proliferating hepatocytes retained their morphologic, phenotypic, and functional characteristics. Using a cell patterning technique, we found that 3T3-J2 fibroblasts stimulate DNA synthesis in hepatocytes by short-range heterotypic cell-cell interactions. When hepatocytes that proliferated in cocultures were harvested and further subcultured either on 3T3-J2 fibroblast feeders or in the collagen sandwich configuration, their behavior was similar to that of freshly isolated hepatocytes. We conclude that adult rat hepatocytes can proliferate in vitro in a coculture cell type-dependent manner, and can be serially propagated by coculturing with 3T3-J2 fibroblasts while maintaining their differentiated characteristics. Our results also suggest that one of the major reasons for the functional differences in hepatocyte cocultures may be due to the different proliferative responses of hepatocytes as a function of coculture cell type. This study provides new insights in the roles of coculture cell types and cell-cell interactions in the modulation of hepatic proliferation and function.     

Expression of a releasable form of annexin II by human keratinocytes

Annexin II is a multifunctional calcium-dependent phospholipid binding protein whose presence in epidermis has previously been reported. However, like other members of annexin family, annexin II has been regarded as either an intracellular protein or associated with the cellular membrane. Here, we report the presence of a releasable annexin II and p11, two monomers of annexin II tetramer, in keratinocyte-conditioned medium (KCM). Proteins present in KCM were fractionated on a gel filtration column and following further evaluation, a releasable protein with apparent MW of 36 kDa was identified. Further characterization identified this protein as the p36 monomer of annexin II tetramer. The phospho-tyrosine antibody did not visualize this protein as the phosphorylated form of p36. Several experiments were conducted to examine whether this protein is soluble or associated with keratinocyte cell membranes in the conditioned medium. A centrifugation of conditioned medium was not able to bring this protein down into the pellet. Surprisingly, the results of Western analysis identified p36 and p11, two monomers of the annexin II tetramer, in conditioned medium derived from either keratinocytes cultured alone or keratinocytes co-cultured with fibroblasts. In contrast to the keratinocyte-conditioned medium in which annexin II was easily detectable, both monomers were barely detectable in conditioned medium collected from dermal fibroblasts. This finding was in contrast to the cell lysates in which p36 was detectable in both keratinocytes and fibroblasts. However, the amount of this protein was markedly higher in keratinocyte lysate relative to that of dermal fibroblasts. Conditioned medium derived from keratinocyte established from adult showed a higher level of annexin II compared to that of keratinocytes established from newborn babies. The expression of p11 seems to increase with differentiation of keratinocytes derived from either adult or newborn skin samples. When the site of annexin synthesis in human skin was examined by immunohistochemical staining, the antibody for p36 localized the annexin to the keratinocyte cell members in the basal and suprabasal keratinocytes. In conclusion, Western blot detection of both p36 and p11 in conditioned medium from skin cells revealed that human keratinocytes, but not fibroblasts, express a releasable monomer form of annexin II which is regulated by differentiation status of keratinocytes. This finding is consistent with the localization of annexin II detected by immunohistochemical staining.     

Effect of 1,25-dihydroxyvitamin D3 on human keratinocytes grown under different culture conditions

Summary 1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂-D₃) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes. The growth and differentiation of keratinocytes in the presence of 1,25-(OH)₂-D₃ using serum-free media formulations has been described previously. This investigation extends these studies to describe various culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of 1,25-(OH)₂-D₃. Keratinocytes were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder layer in keratinocyte growth medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin C-treated dermal human fibroblasts. The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented with growth factors. Keratinocyte cell growth was greatest in the KGM medium. Although the growth of keratinocytes on either feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,25-(OH)₂-D₃. The addition of hydrocortisone and cholera toxin to the medium also affected the response of the keratinocytes to 1,25-(OH)₂-D₃. The antiproliferative effect of 1,25-(OH)₂-D₃ was not altered by varying the extracellular calcium levels from 0.25 to 3 mM. The antiproliferative activity of 1,25-(OH)₂-D₃ is attenuated in cells at low density. Our results suggest that an optimal condition to investigate the ability of 1,25-(OH)₂-D₃ to inhibit keratinocyte proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer. These conditions are not appropriate for investigating the enhancement of differentiation by 1,25-(OH)₂-D₃, but can be used to assay other agents that modulate keratinocyte proliferation. Portions of this work were presented and abstracted at the April 1988 meeting of the Society of Investigative Dermatology (J. Inv. Derm. 90(4): 586; 1988) and the February 1988 meeting of New York Academy of Sciences (Ann NY Acad. Sci. 548: 341–342; 1988).