Keratin polypeptide analysis in fetal and in terminally differentiating newborn mouse epidermis

The keratin polypeptide pattern of neonatal mouse epidermis consists of eight individual polypeptides having molecular weights of between 46,000 and 67,000. Unlike the keratin patterns in adult mouse epidermis, this pattern is not subjects to body site-specific alterations regarding the specific content of distinct polypeptides or the absolute number of keratin constituents. At day 16 of fetal development the neonatal keratin pattern is only partially expressed, it being fully completed just prior to birth at day 19 of gestation. A comparative analysis of the sequential changes in epidermal morphology and keratin pattern during the last third of embryonic development confirms the view that, independent of the species, keratin polypeptides below 60,000 mol. wt. are generated by basal cells, whereas the biosynthesis of high molecular weight keratin members take place in the suprabasal cell compartments of keratinizing epithelia. The site of origin of five polypeptides (60,000, 58,000, 52,000, 49,000, 46,000) could therefore be attributed to the basal cell layer, the remaining three polypeptides (67,000, 64,000, 62,000) being synthesized in the outer metabolically active epidermal layers. Similar conclusions could be drawn after subfractionation of neonatal epidermis into living (basal, spinous, and granular) and dead cell layers (stratum corneum), and investigation of the corresponding keratin patterns. During their progression through the epidermis, two proteins (60,000, 58,000) undergo a hitherto undescribed type of posttranslational modification characterized by a slight increase in size and a change in electrical charge. The mechanism underlying this modification is unknown and it is unclear whether the modification if functional or trivial. The largest keratin polypeptide (67,000) of the protein family -- probably a product of spinous cells -- disappears from the cornified layer without any evidence that it serves as a precursor for smaller keratin subunits.     

The expression of keratin genes in epidermis and cultured epidermal cells

Cultured human epidermal cells and human stratum corneum (callus) contain a number of keratins of different molecular size, but the size distribution is not the same in the two cases. To characterize these keratins in more detail, we compared them by amino acid analysis, immunological reactivity and one-dimensional peptide mapping (Cleveland et al., 1977). No differences in amino acid composition could be detected among keratins of stratum corneum differing in molecular size by as much as 50%, suggesting that some repeating structure may be present in these molecules. Examination of polypeptide fragments produced by partial enzymatic hydrolysis showed strong similarities among all the keratins of stratum corneum and of cultured epidermal cells, even extending to the keratins of rodents; but the keratins of similar size, whether of stratum corneum or cultured cells, were more closely related than keratins of different size. This conclusion was supported by studies of the immunological reactivity of the keratins.How the epidermal cell generates a family of keratins is a problem of considerable interest. The differences in size and structure between the keratins of stratum corneum and cultured epidermal cells suggest that the epidermal cell can modify the expression of its keratin genes.     

Keratin expression in cultures of adult human epidermal cells.

Keratins are complex fibrous proteins characteristic of epithelial cells. We have developed a procedure that allows us to culture and passage adult human dermal keratinocytes in the absence of mesenchymal substrates. Electron microscopic examination of stratifying cultures showed the presence of numerous filament bundles, desmosomes and electron dense granules. The expression of different classes of keratin was examined by immunofluorescence, SDS-PAGE and immunoblots using monoclonal antibodies. The analysis of water-insoluble proteins revealed the presence of keratins of molecular weights 40 Kda, 50-52 Kda, 56 Kda and 65-67 Kda. Our results indicate that the terminal differentiation of keratinocytes may not require dermal factors.     

Keratin filaments of mouse epithelial cells are rapidly affected by epidermal growth factor

The effects of epidermal growth factor (EGF) on the cytokeratin filaments of cultured murine epithelial cells were studied by the indirect immunofluorescence technique with affinity-purified antibodies. Mouse epithelial cells (MMC-E), grown on glass cover slips, and viewed by immunofluorescence microscopy, showed keratin-specific fluorescence as typical bright perinuclear aggregates corresponding to dense paracrystalline granules seen in electron microscopy. Within minutes after an exposure to EGF, the keratin granules in the MMC-E cells decreased. After 10 min of incubation, the cells had spread fibrillar keratin. Such an effect could not be found after a similar exposure to insulin, dexamethasone, dibutyryl cyclic AMP, or antimitotic drugs. EGF, therefore, has a relatively direct effect on the cytoskeletal organization of cultured epithelial cells. These rapid effects on the keratin filaments may explain the simultaneous EGF-induced ultrastructural surface changes of the cells. EGF may thus function as a regulatory factor in the migration of epithelial cells and in the mobility of their cell membranes. The epithelial cell line, MMC-E, should prove a useful model for studies on the action of EGF on nontransformed epithelial cells in vitro.     

Cloning and expression of human islet amyloid polypeptide in cultured cells

Efforts to clone amyloidogenic proteins in the cells often have resulted in cell death. We report successful cloning and expression of recombinant human islet amyloid polypeptide (hIAPP) in cultured mammalian cells. Amylin gets secreted, forms fibrils that are toxic to target cells like beta cells of rat and human. The study involves cloning of full-length amylin in fluorescent protein vector followed by transfection into mammalian cells. The transfected cells with recombinant human amylin, secrete the translated protein corresponding to 37-amino acid native mature IAPP. The mature IAPP secreted out of the cell is purified and characterized by MALDI-TOF/TOF-MS and Western blotting. Purified IAPP forms fibrils as seen by Thioflavin-T fluorescence and AFM, and these fibrils were cytotoxic towards pancreatic cell line RIN5mf cells.     

Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins

Monospecific antibodies to mouse epidermal keratins were generated in rabbits and guinea pigs by injecting synthetic peptides of unique keratin sequences. The sequences were deduced from nucleotide sequences of cDNA clones representing basal (K14) and suprabasal (K1 and K10) cell-specific and hyperproliferative (K6) keratins of both the type-I and type-II subclasses. By applying single-and double-label immunofluorescence analysis, the expression of keratin peptides was analyzed in cultured keratinocytes maintained in the basal or suprabasal cell phenotypes. These cell types were selected by growth in medium containing 0.05 mM Ca2+ (basal cell) or 1.4 mM Ca2+ (suprabasal cell). The cultured basal cells expressed K6 and K14, but less than 1% expressed K1 and K10. Within a few hours after being placed in 1.4 mM Ca2+, K1 expression was observed, and by 24 h, 10%-17% of the cells expressed K1. K10 expression appeared to lag behind K1 expression, with only 5%-10% of cells in 1.4 mM Ca2+ exhibiting K10 immunoreactivity. Double-labeling studies indicated that virtually all K10-positive cells also expressed K1, while only about one-half of the K1-positive cells expressed K10. The treatment of basal cells with retinoic acid at pharmacological concentrations prevented the expression of K1 and K10 when cells were challenged by 1.4 mM Ca2+. Similarly, the introduction of the v-rasH oncogene into basal cells by a defective retroviral vector prevented the expression of suprabasal keratins in 1.4 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Keratin filaments of cultured human epidermal cells. Formation of intermolecular disulfide bonds during terminal differentiation.

Human epidermal cells grown in culture synthesize abundant keratins. These keratins are similar to those of stratum corneum of human epidermal callus in their insolubility in dilute aqueous buffers, their molecular weight range of 40,000 to 60,000, their immunolgical reactivity, and their ability to assemble into 80 A tonofilaments in vitro; but there are differences in the molecular weights of some of the proteins, the number of components, and their charge heterogeneity, related at least in part to phosphorylation. About 30% of all the proteins of living cultured keratinocytes consists of keratins, compared with over 85% of stratum corneum. All the keratins of human stratum corneum were found to be cross-linked by intermolecular disulfide bonds while most keratins of the living cells were not. As the cells mature in Methocel-stabilized suspension culture, their keratins become increasingly disulfide cross-linked. When uncross-linked tonofilaments of living keratinocytes are dissolved in 8 M urea and the filaments reconstituted in vitro their keratins become disulfide cross-linked under aerobic conditions and consequently insoluble in solutions of 8 M urea or sodium dodecyl sulfate. The results indicate that the uncross-linked state of the keratins in living cells is due to the reducing intracellular environment and not to a precursor state related to the primary structure of the proteins. The disulfide cross-links stabilizing the keratin filaments must be distinguished from the epsilon-(gamma-glutamyl)lysine cross-links stabilizing the cornified cell envelope.     

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.     

Regulated expression of human atrial natriuretic polypeptide gene in mouse L cells

To investigate whether the human atrial natriuretic polypeptide (hANP) gene is responsive to glucocorticoid, we co-introduced the hANP gene (with SV40 enhancer) with HSV-tk gene into mouse tk- L cells. The transformants with hANP gene with SV40 enhancer expressed hANP specific RNAs. The administration of 1 microM dexamethasone reduced the expressed hANP specific RNAs, especially those that had a physiological initiation site. These results suggest that the hANP gene is really a glucocorticoid responsive gene and may be negatively regulated by glucocorticoid.     

Growth kinetics in newborn mouse epidermis: response to epidermal chalone.

Epidermal DNA synthesis, the epidermal mitotic rate, and the responsiveness to the epidermal G1 and G2 inhibitors were examined in newborn mice at different times after birth. The rate of epidermal cell renewal was in general low during the first two weeks of life. Later the two growth parameters increased and reached very high values at 32-33 days after birth. The rate of epidermal cell proliferation then decreased to a level comparable with that found in adult hairless mouse epidermis at 40-45 days. A single i.p. injection of skin extract containing the two epidermal growth inhibitors induced varying types of responses. The epidermal G2 inhibitor stimulated the mitotic rate on day 2 and day 10, but inhibited it on all other days. The epidermal G1 inhibitor brought about an increase in epidermal DNA synthesis on day 6 and possibly on the following days. No response at all seen at 2, 4, 17, and 32 days after birth. At the other examined times the inhibition was similar to that found in adult mice. These findings differed from those made in vitro on separated newborn mouse epidermal cells (our own unpublished data), and we suggest that the variability of newborn mouse epidermis could be an expression of the immaturity of the skin as a whole, and that dermis in some way modifies the response of epidermis to exogenous epidermal chalone. Our study did not support the theory that the nonresponsiveness of newborn mouse epidermal at certain times could be due to the presence of nonresponsive stem cells in epidermis.     

Enhancement of melanotic expression in cultured mouse melanoma cells by retinoids

Retinoic acid (RA), which reduces the rate of cell proliferation in S91 mouse melanoma clone C2 cells, was found to stimulate the expression of their melanotic phenotype. RA treatment also induced the extension of long cellular processes. The RA effects on melanogenesis included stimulation of tyrosinase activity and augmentation of cellular melanin content to levels 3- to 4-fold higher than in untreated cultures at similar cell densities. These effects became apparent after 48 hours of exposure to 10(-5) M RA and increased thereafter. Half-maximal stimulation in cells treated for 6 days occurred at 5 X 10(-7) M RA. Although the degrees of melanogenesis enhancement by RA (10(-5) M) and by alpha-melanocyte stimulatory hormone (2 X 10(-7) M) were similar, the former did not alter the intracellular cAMP level, whereas the latter induced a transient 4-fold increase. In high-passage (p28) cells, as well as in low-passage cells (less than p10) treated with tyrosinase inhibitor phenylthiocarbamate, melanin synthesis was suppressed in the absence and presence of RA, yet the ability of RA to inhibit cell proliferation was not compromised. In the presence of the tumor promotor phorbol myristate acetate (greater than 5 X 10(-9) M) melanin synthesis in control as well as in cells exposed to RA was dramatically inhibited. Phorbol which is not active in tumor promotion had no effect on melanogenesis. In addition to RA, other retinoids, such as 13-cis-retinoic acid, retinyl acetate, the TMMP analog of RA and the phenyl analog of RA, but not the pyridyl analog of RA or retinyl palmitate, also inhibited cell growth and enhanced melanin synthesis.     

Microflowfluorometric evaluation of homeostasis in cultured epidermal cells

In isolating and culturing in vitro populations of basal cells from adult guinea pig skin, it has been possible to show that these cells are sensitive to both G1 and G2 inhibitions. Only a small fraction (10 percent or less) of the G1 blocked cell population would be governed by G1 inhibitory messages released by suprabasal, maturing keratinocytes. As regards the G2 block in vitro experiments confirm that basal cells produce a G2 blocker to which about 9 per cent or less are susceptible. In conclusion basal cells in culture are sensitive to homeostatic regulation as in vivo.     

Rhythmic autocrine activity in cultured insect epidermal cells

It is now well established that ecdysteroids can be produced in insects in the absence of prothoracic glands. In this respect, it has been shown that cells in culture can produce ecdysteroids. Our aims were: (1) to determine whether ecdysteroid target cells of epidermal origin could also be the source of ecdysteroids; (2) to monitor more accurately the kinetics of ecdysteroid production; and (3) to check for possible relationships between this synthetic activity and dynamics of cell division. An insect cell line (IAL-PID2) established from imaginal discs of the Indian meal moth, Plodia interpunctella, with wild-type sensitivity to ecdysteroids was used in our study. Our results showed that the Plodia cell line exhibited autocrine activity. When division of IAL-PID2 cells was synchronized, a rhythmic production of ecdysteroids was observed. However, further experiments indicated that this rhythmicity could be cell autonomous. This led us to anticipate the existence of two cell subpopulations that would be able to produce ecdysteroids rhythmically, a minor one that would be cell cycle serum-independent population, and a major population that would need serum growth factors to proliferate and produce ecdysteroids. Qualitative study of the ecdysteroid content of the media clearly showed that ecdysone was the major immunoreactive product. Taken together, our findings clearly show that an insect cell line of epidermal origin is capable of rhythmic autocrine production of ecdysteroids. These results support the hypothesis that alternate sites for ecdysteroid production in vivo may exist and could play a role in local regulation of development. We now plan to determine the cellular basis of this rhythmic autocrine activity and to confirm the existence of growth factor-autonomous cells in the culture as well as the potent role played by ecdysteroids in the cross-talk between various cell subpopulations.     

Biosynthesis of islet amyloid polypeptide. Elevated expression in mouse beta TC3 cells

Islet amyloid polypeptide (IAPP) messenger RNA levels, biosynthesis, processing, and secretion were studied in cultured mouse beta TC3 insulinoma cells. Northern blot analysis revealed that the size of IAPP mRNA (0.9 kb) in beta TC3 cells was the same as that in normal mouse islets; IAPP mRNA was approximately 60% of the level of insulin mRNA in beta TC3 cells. However, the ratio of synthesis of insulin to IAPP was approximately 6:1, suggesting that IAPP mRNA is not translated efficiently in these cells. Metabolic labeling of beta TC3 cells with [3H]leucine revealed the synthesis of both a precursor form of IAPP (pro-IAPP) of apparent Mr 7400 and a mature form (IAPP) of apparent Mr 3900. In pulse-chase experiments, pro-IAPP could be shown to be processed to IAPP in a manner similar to proinsulin. The t1/2 for conversion of pro-IAPP to IAPP was about 25 min, faster than the t1/2 for proinsulin to insulin of 70 min. A significant proportion of newly synthesized IAPP and insulin precursors were secreted via a constitutive pathway from beta TC3 cells. Possible effects of dexamethasone and forskolin on IAPP mRNA levels and biosynthesis were examined but no effects were observed. In conclusion, the IAPP gene is strongly expressed in beta TC3 cells leading to the biosynthesis, proteolytic processing, and secretion of IAPP, a putative islet hormone.