Aromatic hydrocarbons upregulate glyceraldehyde-3-phosphate dehydrogenase and induce changes in actin cytoskeleton. Role of the aryl hydrocarbon receptor (AhR).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme involved in several cellular functions including glycolysis, membrane transport, microtubule assembly, DNA replication and repair, nuclear RNA export, apoptosis, and the detection of nitric oxide stress. Therefore, modifications in the regulatory ability and function of GAPDH may alter cellular homeostasis. We report here that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone, which are well-known ligands for the aryl hydrocarbon receptor (AhR), increase GAPDH mRNA levels in vivo and in vitro, respectively. These compounds fail to induce GAPDH transcription in an AhR-null mouse model, suggesting that the increase in GAPDH level is dependent upon AhR activation. To analyse the consequences of AhR ligands on GAPDH function, mice were treated with TCDD and the level of liver activity of GAPDH was determined. The results showed that TCDD treatment increased GAPDH activity. On the other hand, treatment of Hepa-1 cells with beta-naphthoflavone leads to an increase in microfilament density when compared to untreated cultures. Collectively, these results suggest that AhR ligands, such as polycyclic hydrocarbons, can modify GAPDH expression and, therefore, have the potential to alter the multiple functions of this enzyme.

Functional domains of human growth hormone necessary for the adipogenic activity of hGH/hPL chimeric molecules.

Genetic analysis through construction of chimeric genes and their transfection in mammalian cells could provide a better understanding of biological functions of native or modified proteins, and would allow the design of new gene constructs encoding peptides that mimic or block ligand interaction with target tissues. To identify the hGH domains responsible for induction of adipose differentiation we constructed hGH/hPL chimeric molecules using homologous DNA mutagenesis, since hGH, but not human placental lactogen (hPL), promotes adipose differentiation in mouse 3T3-F442A cells. We assayed their adipogenic activity in an autocrine/paracrine biological model consisting of transiently transfected 3T3-F442A cells with the chimeric constructs. Plasmid DNAs carrying these constructs were transfected into growing 3T3-F442A cells, and cultures were further maintained for 7 days to differentiate into adipocytes. Secretion of transfected hGH/hPL chimeric proteins into the medium was in the range of 5-25 ng/ml. Adipogenic activity was a property only of those chimeric proteins that contained hGH exon III together with either hGH exon II or hGH IV. Our results also suggest that hGH binding site-2 is composed of two structural subdomains: subsite 2A encoded by exon II of hGH and subsite-2B encoded by exon IV. We also suggest that full adipogenic activity requires the presence of binding site-1 and any of the subsites of binding site-2. This simple autocrine/paracrine biological model of gene transfection allows the analysis of specific biological activity of products encoded by modified genes.

Frozen cultured sheets of human epidermal keratinocytes enhance healing of full-thickness wounds in mice.

Sheets of cultured allogeneic human keratinocytes have been used for the treatment of burns and chronic leg ulcers but there has been no animal assay for the therapeutic action of these cultures. In order to analyze the effects of frozen cultures of human keratinocytes on wound healing, we have developed such an assay based on the rate of repair of full-thickness skin wounds in immunocompetent NMR1 mice. Reepithelialization of the control wounds, originating from the murine epithelium at the edge of the wound, occurred at a constant rate of advance of 150 microm/day. When frozen cultured human epidermal sheets were thawed at room temperature for 5-10 min and applied to the surface of the wound, the murine epithelium advanced at 267 microm/day. Most wounds treated with frozen cultures completely healed after 10 days, whereas most control wounds required 16 days. The accelerated reepithelialization did not depend on the presence of proliferative human keratinocytes in the frozen cultures. The cultures also promoted early formation of granulation tissue and laminin deposition over the surface of the wound bed. This simple assay should permit quantitative analysis of the effects on healing exerted not only by cultured cells, but also by proteins and small molecules.

Cultivation of rabbit corneal epithelial cells in serum-free medium.

PURPOSE: To establish conditions for cultivation, serial growth, and normal differentiation of corneal epithelial cells in serum-free medium (SFM). METHODS: Rabbit corneal epithelial cells were co-cultured with lethally treated 3T3-cell feeder layers. Instead of serum, medium was supplemented with serum albumin, hormones, and other additives. Cell growth was quantitated spectrophotometrically with a new rhodamine-B staining protocol with a sensitivity range of 5 X 10(3) to 1 x 10(5) cells/cm2. Keratin expression was analyzed by immunostaining or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. RESULTS: In SFM, without growth factors, cells grew no more than six to eight doublings, but when 10 ng/ml epidermal growth factor were added, serial transfer was possible, and epithelial cells grew to up to 18 to 20 doublings (three cell passages). Two cell colony types were seen: One type was composed of nonstratified proliferating cells, and the other of stratified cells expressing high levels of the differentiation-linked keratins K3 and K12. Confluent cultures formed a four- to five-layer stratified epithelium whose suprabasal cells were stained with anti-K12 antiserum. Acidic and basic fibroblast growth factors and epidermal growth factor reduced the expression of keratins K3 and K12. Transforming growth factor-alpha and epidermal growth factor led to the highest stimulation of cell proliferation. Limbal, peripheral, and central corneal epithelial cells showed similar clonal growth abilities, but colony size was larger for cells derived from limbal epithelium. CONCLUSIONS: These SFM conditions support the serial transfer, normal differentiation, and formation of typical corneal epithelium by cultured corneal epithelial cells and are useful in studying and assaying a variety of cytokines and compounds that modulate corneal epithelial cell proliferation and differentiation.

Cultivation, serial transfer, and differentiation of epidermal keratinocytes in serum-free medium.

We describe serum-free culture conditions for human epidermal keratinocytes using lethally treated 3T3 cells as feeder layers and normal Ca++ concentrations (1.2 mM), in a DMEM/F12-Ham nutrient mixture supplemented with several additives, and 10 mg/ml bovine serum albumin instead of animal serum. Keratinocytes were serially grown to 15-18 cell generations (4 subcultivations) and formed a stratified squamous epithelium that could be detached as a graftable epithelial sheet. EGF and TGF alpha significantly increased keratinocyte proliferation under these conditions; EGF reduced the expression of keratin K1, which is specific for stratified and terminally differentiated epidermal keratinocytes. In contrast with previous reports, the serum-free medium we describe here supports serial growth and normal differentiation of human epidermal keratinocytes, and the formation of graftable stratified epithelia; it also supports the assay of a variety of cytokines or compounds that modulate epidermal keratinocyte proliferation and differentiation.

Regulation of K3 keratin gene transcription by Sp1 and AP-2 in differentiating rabbit corneal epithelial cells.

Rabbit corneal epithelial cells cultured in the presence of 3T3 feeder cells undergo biochemical differentiation, as evidenced by their initial expression of K5 and K14 keratins characteristic of basal keratinocytes, followed by the subsequent expression of K3 and K12 keratin markers of corneal epithelial differentiation. Previous data established that mutations of an Sp1 site in a DNA element, E, that contains overlapping Sp1 and AP-2 motifs reduce K3 gene promoter activity by 70% in transfection assays. We show here that Sp1 activates while AP-2 represses the K3 promoter. Although undifferentiated corneal epithelial basal cells express equal amounts of Sp1 and AP-2 DNA-binding activities, the differentiated cells down-regulate their Sp1 activity slightly but their AP-2 activity drastically, thus resulting in a six- to sevenfold increase in the Sp1/AP-2 ratio. This change coincides with the activation and suppression of the differentiation-related K3 gene and the basal cell-related K14 keratin gene, respectively. In addition, we show that polyamines, which are present in a high concentration in proliferating basal keratinocytes, can inhibit the binding of Sp1 to its cognate binding motif but not that of AP-2. These results suggest that the relatively low Sp1/AP-2 ratio as well as the polyamine-mediated inhibition of Sp1 binding to the E motif may account, in part, for the suppression of the K3 gene in corneal epithelial basal cells, while the elevated Sp1/AP-2 ratio may be involved in activating the K3 gene in differentiated corneal epithelial cells. Coupled with the previous demonstration that AP-2 activates the K14 gene in basal cells, the switch of the Sp1/AP-2 ratio during corneal epithelial differentiation may play a role in the reciprocal expression of the K3 and K14 genes in the basal and suprabasal cell layers.

Combined use of allograft and autograft epidermal cultures in therapy of burns.

Cultivation of human epidermal keratinocytes made possible the use of cultured autografts as part of the therapy of extensively burned patients. On the basis of our early results using banked cultured allografts and autografts, we developed an integral and combined burn therapy comprising banked cultured allografts for rapid healing of skin donor sites and deep partial-thickness burns, conventional split-thickness skin autografting, and when needed, cultured autografts for full-thickness burns. We compared hospital stay in 32 burn patients treated with the combined therapy and in 39 who were not treated with cultured epidermis. Three groups of patients were defined: 15 to 29 percent (n = 12), 30 to 49 percent (n = 10), and more than 49 percent (n = 10) burned body surface area. We found a 20 to 29 percent decrease in hospital stay in patients with up to 49 percent burned body surface area and a 46 percent reduction in patients suffering more extensive burns. Survival rate of extensively burned patients also was increased. We took advantage of the availability of banked cultured allografts for ambulatory treatment, without hospitalization, of pediatric patients with 5 to 20 percent burned body surface area. We show for the first time the use and benefits of this combined therapy.

22-kDa and 20-kDa hGH isoforms show differential effects when assayed in 3T3-F442A and 3T3-F442A/C4 adipocytes.

To known whether 22.0-kDa human growth hormone (hGH22K) and the 20.0-kDa isoform (hGH20K) show different activities, we used 3T3-F442A and 3T3-F442A/C4 cells to evaluate their adipogenic and metabolic effects. Both isoforms had similar adipogenic and insulin-like activities. Lipolytic and diabetogenic effects of hGH22K were, respectively, 12.5 and 1.7-fold higher than those found for hGH20K. The 3T3-F442A/C4 clone was not responsive to insulin-like and diabetogenic effects of hGH. The results suggest that adipogenic and lipolytic effects of hGH are mediated by mechanisms different from those involved in insulin-like and diabetogenic activities.

A preadipose 3T3 cell variant highly sensitive to adipogenic factors and to human growth hormone.

We describe a new Swiss 3T3 preadipose clone, 3T3-F442A/C4, which shows higher sensitivity to serum adipogenic factors and to human growth hormone as compared to other 3T3 preadipose clones. The 3T3-F442A/C4 clone exhibited several characteristics different from the parental 3T3-F442A cells, mainly a high extent of adipose conversion under culture conditions that are non-adipogenic for the parental cells. The 3T3-F442A/C4 cells are not committed to undergo adipose differentiation, since they do not differentiate into adipocytes under serum-free or low-serum culture conditions, unless adipogenic factors or growth hormone are added into the culture medium. The 3T3-F442A/C4 cells showed 1.5- to 3.6-fold higher sensitivity to serum adipogenic factors and 5- to 6-fold higher sensitivity to human growth hormone as compared to the 3T3-F442A cells. The 3T3-F442A/C4 variant clone also differed from the parental clone by having a shorter population doubling time, an increased saturation density, and lower activity levels in some biochemical markers of adipose differentiation. On the other hand, the new variant clone has a similar proportion of cells susceptible to become adipocytes, and a similar response to insulin as compared to the parental cells. Our results show that the 3T3-F442A/C4 cells represent a new 3T3 preadipose clone that could be useful as a bioassay to evaluate growth hormone activity, as well as to purify and characterize hormones, adipogenic factors, and those compounds that affect mammalian adipogenesis.

Inhibition of 3T3 adipogenesis by retinoic acid is not mediated by cytoplasmic retinoic acid-binding protein.

Retinoic acid (RA) inhibits 3T3 adipogenesis in a dose-dependent and reversible manner, but its mechanism of action remains unknown. 3T3-F442A cell variants obtained by mutagenesis with nitrosoguanidine and/or selection with high RA concentrations showed different resistance to RA cytotoxicity and underwent adipose conversion of various extents when they were cultured in adipogenic conditions. Commitment to adipose differentiation was also inhibited by RA in these clones. Gel filtration chromatography showed the presence of a cytosolic RA-binding activity in the parental cells but not in three of the variant clones isolated. We demonstrate that cytoplasmic RA-binding activity is not essential for the inhibitory effects of the retinoid on 3T3 adipogenesis, or for resistance to RA cytotoxicity. Other mechanisms should be involved to explain the inhibition of adipose differentiation by RA.

Development of a spontaneous permanent cell line of rabbit corneal epithelial cells that undergoes sequential stages of differentiation in cell culture.

Established epithelial cell lines that retain their differentiation potential and growth regulatory characteristics can provide valuable tools for studying gene regulation, extracellular matrix synthesis or growth factor response. They are also useful for drug development and toxicity testing. Experiments were therefore carried out to optimize culture conditions for the long-term, serial transfer of corneal epithelial cells in the presence of 3T3 feeder layers; and to establish a permanent cell line. In such experiments, rabbit corneal epithelial cells were seeded at low inoculation densities, and transferred every 5 days. After 80 population doublings, an epithelial cell line, RCE1, emerged. The cell line is heteroploid, with an average population doubling time of 15.5 hours (vs 18 hours for primary cultures). When RCE1 cells reached confluence, they stratified to form a three- to five-layered epithelium and expressed the differentiation-related keratin pair K3/K12 as shown by immunoblot and immunostaining. Biosynthetic labeling of proliferating, confluent and stratified cultures further showed that RCE1 cells expressed keratin pairs K5/K14, K6/K16 and K3/K12, thus mimicking faithfully the stage-dependent differentiation of primary cultures of rabbit corneal keratinocytes. The results demonstrated that RCE1 cells provide a useful model for studying corneal cell growth and differentiation.

HGH isoforms: cDNA expression, adipogenic activity and production in cell culture.

We have isolated, cloned and achieved functional expression of the cDNAs for both 22 kDa and 20 kDa human growth hormone (hGH) isoforms. A selective cDNA cloning strategy was used to preferentially and simultaneously obtain both hGH 22 kDa and hGH 20 kDa cDNAs. These were used to construct minigenes which were subcloned into two eukaryotic expression vectors and then introduced transiently in COS-7 cells and stably into CHO cells in culture. Transfection assays in COS-7 cells of both minigenes allowed the detection of the secreted hGH 22 kDa and hGH 20 kDa. These hGHs isoforms secreted into COS-7 medium were able to specifically promote differentiation of 3T3-F442A preadipocytes to adipose cells. Adipocyte differentiation was quantitated by Oil Red O triacylglycerol staining or glycerophosphate dehydrogenase activity. Furthermore, stable CHO cell lines have been derived that produce these hGH isoforms.

Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O.

Cultured 3T3-F442A cells differentiate into adipocytes and accumulate lipid droplets in the cytoplasm. When fat cells are stained with Oil red O, the degree of staining seems to be proportional to the extent of cell differentiation. We report here a fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm. The results show that Oil red O specifically stains triglycerides and cholesteryl oleate but no other lipids. This technique is a valuable tool for processing large numbers of cell cultures or samples in which adipose differentiation and/or accumulated triglycerides is to be quantitated.

Commitment of adipocyte differentiation in 3T3 cells is inhibited by retinoic acid, and the expression of lipogenic enzymes is modulated through cytoskeleton stabilization.

Retinoic acid (RA), at 1-10 microM, inhibited adipose conversion of 3T3-F442A cells as determined by the activities of lipogenic enzymes, glycerophosphate dehydrogenase (GPD) and malic enzyme. This inhibition was reversible by RA removal, but the increase in lipogenic enzyme activities was considerably delayed in a dose-dependent manner. The onset of the two lipogenic enzyme activities could be regulated somewhat independently, suggesting that expression of the two enzymes is subject to noncoordinated regulation. The RA-inhibited cells had a more flattened and elongated shape, suggesting cytoskeletal changes. Cytochalasin B (CB) did not prevent RA inhibition and did not promote adipose conversion in cultures supplemented with nonadipogenic medium. Reversion of inhibition was accelerated if cells were cultured for 3 days with adipogenic medium containing CB. The drug promoted an early increase in lipogenic enzyme activities. On the other hand, cells cultured on fibronectin-coated dishes, a condition that stabilizes actin cytoskeleton, do not undergo adipocyte differentiation. However, we show here that cells cultured on fibronectin and changed to nonadipogenic medium containing insulin underwent adipose conversion; in contrast, cells treated with RA and then supplemented with nonadipogenic medium containing insulin, but without the retinoid, did not undergo differentiation. We conclude that RA blocks adipose conversion probably before commitment to differentiation, and modulates lipogenic enzyme expression in a noncoordinated manner through changes in cytoskeletal elements, whereas fibronectin blocks phenotype expression in differentiating cells.