Nancy L. R. Bucher

The differentiation of 3T3 preadipocytes into adipocytes is accompanied by a transient induction of C/EBPbeta and C/EBPdelta expression in response to treatment of the cells with methylisobutylxanthine (MIX) and dexamethasone (DEX), respectively. In this report, we demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) expression in 3T3-L1 preadipocytes is induced by MIX and DEX, suggesting that C/EBPbeta and C/EBPdelta may be involved in this process. Using a tetracycline-responsive expression system, we have recently shown that the conditional ectopic expression of C/EBPbeta in NIH 3T3 fibroblasts (beta2 cells) in the presence of DEX activates the synthesis of peroxisome PPARgamma mRNA. Subsequent exposure of these cells to PPAR activators stimulates their conversion into adipocytes; however, neither the expression of C/EBPbeta nor exposure to DEX alone is capable of inducing PPARgamma expression in the beta2 cell line. We find that unlike the case for 3T3 preadipocytes, C/EBPdelta is not induced by DEX in these 3T3 fibroblasts and therefore is not relaying the effect of this glucocorticoid to the PPARgamma gene. To define the role of glucocorticoids in regulating PPARgamma expression and the possible involvement of C/EBPdelta, we have established an additional set of NIH 3T3 cell lines expressing either C/EBPdelta alone (delta23 cells) or C/EBPdelta and C/EBPbeta together (beta/delta39 cells), using the tetracycline-responsive system. Culture of these cells in tetracycline-deficient medium containing DEX, MIX, insulin, and fetal bovine serum shows that the beta/delta39 cells express PPARgamma and aP2 mRNAs at levels that are almost equivalent to those observed in fully differentiated 3T3-L1 adipocytes. These levels are approximately threefold higher than their levels of expression in the beta2 cells. Despite the fact that these beta/delta39 cells produce abundant amounts of C/EBPbeta and C/EBPdelta (in the absence of tetracycline), they still require glucocorticoids to attain maximum expression of PPARgamma mRNA. Furthermore, the induction of PPARgamma mRNA by exposure of these cells to DEX occurs in the absence of ongoing protein synthesis. The delta23 cells, on the other hand, are not capable of activating PPARgamma gene expression when exposed to the same adipogenic inducers. Finally, attenuation of ectopic C/EBPbeta production at various stages during the differentiation process results in a concomitant inhibition of PPARgamma and the adipogenic program. These data strongly suggest that the induction of PPARgamma gene expression in multipotential mesenchymal stem cells (NIH 3T3 fibroblasts) is dependent on elevated levels of C/EBPbeta throughout the differentiation process, as well as an initial exposure to glucocorticoids. C/EBPdelta may function by synergizing with C/EBPbeta to enhance the level of PPARgamma expression.

Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes.

Freshly isolated adult rat hepatocytes exhibit a flat, extended morphology when cultured on dried rat tail collagen in the presence of growth factors; they actively synthesize DNA and express high levels of cytoskeletal mRNAs and proteins (actin, tubulin, cytokeratins, vinculin, alpha-actinin, and desmoplakin), while exhibiting low levels of liver-specific mRNAs (albumin, alpha 1-inhibitor III, and alpha 1-antitrypsin) and limited synthesis and secretion of albumin. Hepatocytes cultured on hydrated gel matrix from the Engelbreth-Holm-Swarm (EHS) mouse tumor form small spherical aggregates and exhibit low DNA, cytoskeletal mRNA, and protein synthesis, while at the same time exhibiting elevated liver-specific mRNAs and albumin production; these cells, therefore, more nearly conform to the program of gene expression seen within the normal animal. Hepatocytes on hydrated rat tail collagen resemble those on dry collagen when cultured at low density, but at high density they form compact trabecular aggregates, synthesize negligible amounts of DNA, and maintain a pattern of gene expression resembling that of hepatocytes seeded on the EHS matrix. If cell morphology is compact, as on EHS or on hydrated rat tail collagen when densely populated, DNA synthesis and expression of cytoskeletal genes are low, while liver-specific mRNAs are abundant. When cells are extended the opposite is the case. Without the growth supplement DNA synthesis is low throughout but gene expression is little affected. These studies point to the importance of cell-cell and cell-matrix interactions in determining the differentiated phenotype of hepatocytes, and they reveal an inverse relationship between cytoskeletal and liver-specific protein expression.

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.