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Growth hormone and adipose differentiation : Growth hormone-induced antimitogenic state in 3 T 3F 442 A preadipose cells ( terminal differentiation / hormonal pathway )
| Content Provider | Semantic Scholar |
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| Author | Corin, Robert E. Guller, Seth Kai-Yuan, Wu Sonenberg, Martin |
| Abstract | An additional activity for pltuitary growth hormone is described-i.e., the in vitro Induction of an andtimitogenic state in murine 3T3-F442A preadipocyte fibroblasts. We previously developed a serum-free, hormonally defined medium permissive for the adipose differentiation of 3T3F442A cells. When 3T3-F442A fibroblasts were maitined in serum-fr medium without insulin but with growth hormone (2 nM), typical adipose differentiation did not occur. However, we found that growth hormone induced a state of cellular refractoriness to the mitogenic stimulus of fetal bovine serum as assayed by de novo DNA synthesis. The mitogen reactory condition (i.e., the antimitogenic state) was time-dependent (half maximal at -2.5 days) and growth hormone concentration-dependent (half maximal and maximal at ".0.05 and 2.0 nM, respectively). The antimitogenic state was siy induced by growth hormone and was not mediated by insulinlike growth factor I or prolactin. The growth hormone-Induced antimitogenic state was completely reverib. The antimitogenic state was not induced by growth hormone in 3T3-C2 cells, a sister clone of 3T3 cells that exhibits essentially no adipose conversion. The kinetics for growth hormone-dependent commitment to adipose differentiation and induction of the antimitogenic state were similar. We suggest a relationship of growth hormone-induced antimitogenic state and the growth hormone-induced adipose differentiation of 3T3-F442A cells. Murine 3T3-F442A preadipocytes are immortal embryonic fibroblastic cells ofmesenchymal origin (1). By tissue culture criteria, these cells are not transformed since the cells are highly contact inhibited during growth, require high concentrations of animal serum to grow, and are unable to grow in suspension culture (1). Growing cells exhibit no differentiated phenotypes. When 3T3-F442A cells achieve quiescence, serum factors are able to induce terminal adipose differentiation (2-4). The physiological relevance of this phenomenon is emphasized by the finding that subcutaneous injection of 3T3-F442A fibroblasts into nude mice yields fully developed fat pad tissue at the sites of injection (5). Green and coworkers (6, 7) identified pituitary growth hormone (GH) as one of the adipogenic factors of serum. Adipose differentiation is under multihormone control as demonstrated by a number ofgroups (4, 7-10). We developed a serum-free, hormonally defined medium that is permissive for terminal adipose differentiation of 3T3-F442A fibroblasts (4). We found that both GH and insulin were required for adipose differentiation of 3T3-F442A cells (4). We concluded that the insulin requirement was mediated by the insulin receptor since insulin-like growth factor I (IGF-I) could not substitute for insulin even at micromolar concentrations (4). GH did not induce any of the well-documented adipose phenotypes [e.g., high specific activity for the lipogenic marker enzyme glycerol-3-phosphate dehydrogenase (GPDH), development offat inclusions, lipogenic and lipolytic hormonal responsiveness, and loss of proliferative capacity (1-13)]. However, GH did induce a distinct phenotype we designated the GH-primed phase ofthe adipose differentiation program (8, 11). Primed cells have entered a GH-induced, insulin-sensitive, and differentiation-permissive condition (8, 11). This is a regulated event in which quiescent cells become susceptible to GH. It is our hypothesis that the primed state is a differentiation-permissive quiescent state that has unique characteristics and determinants. When compared to quiescent cells that are not exposed to GH, primed cells exhibit a number of distinct features, including altered macromolecular synthesis, morphology, and mitogen responsiveness (8, 12). The primed state of 3T3-F442A preadipocytes is reversible in that all cells resume fibroblastic appearance and behavior when diluted to subconfluent density in a growth-permissive medium; i.e., they return to the typical growing fibroblastic cell state (4, 8, 11). 3T3-F442A preadipocytes that have been GH-primed in our serum-free medium are in a permissive condition for expression of the adipose program (4, 8). Insulin is required for cells to enter the expression phase of adipose differentiation (4, 8). Zezulak and Green (10) demonstrated that one of the functions ofGH in 3T3-F442A cell differentiation was to endow newly formed adipocytes with IGF-I responsiveness not seen in undifferentiated cultures at confluence. IGF-I then induces a self-limited growth and division (two to four divisions) of fat cells [i.e., clonal expansion (13)]. We found that 3T3-F442A adipocytes generated in our serum-free medium did not clonally expand. This indicated that a serum factor(s), in addition to GH and IGF-I, was required for expansion (4, 11). Our previous observation that GH-primed quiescent 3T3F442A preadipocytes exhibited diminished DNA synthesis in response to human platelet-derived growth factor (PDGF) and insulin (8) led us to further characterize this phenomenon. Cells exposed toGH for 3 days exhibited nearly 50% decreasedDNA synthesis in response to insulin and PDGF in serum-free medium. However, the importance of this phenomenon was unclear since PDGF and insulin are weakly mitogenic for growtharrested 3T3-F442A cells and do not have the ability to support cell growth and division (R.E.C., unpublished observations; ref. 8). In the present study, we found that 3T3-F442A cells in serum-free medium developed a GH-dependent refractoriness to the potent growth signals of fetal bovine serum (FBS) (i.e., an antimitogenic state). The GH-induced antimitogenic state was characterized and its significance for the role of GH in Abbreviations: GH, growth hormone; Met-hGH, recombinant human growth hormone; PDGF, human platelet-derived growth factor; IGF-I, human insulin-like growth factor I; FBS, fetal bovine serum; GPDH, glycerol-3-phosphate dehydrogenase. tTo whom reprint requests should be addressed at: Memorial SloanKettering Cancer Center, Box 121, 1275 York Avenue, New York, NY 10021. 7507 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ยง1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 87 (1990) differentiation is discussed within the context of a cell cycle model for adipose differentiation. MATERIALS AND METHODS The composition of serum-free medium and the sources of the components were previously described (4). Recombinant human growth hormone (Met-hGH) was a gift from Genentech and Eli Lilly, bovine prolactin was from the National Pituitary Program (lot AFP-6300), human recombinant IGF-I was a gift from Daniel Burleigh, Jr., of International Minerals and Chemicals (Northbrook, IL), bovine GH was purified in our laboratory (14), culture-grade PDGF was from Collaborative Research, [3H]thymidine (specific activity = 20 Ci/ mmol; 1 Ci = 37 GBq)' was from New England Nuclear, calf serum was from GIBCO, and FBS was from HyClone. 3T3-F442A and 3T3-C2 cells were generously provided by Howard Green of Harvard University. Growth, 'maintenance, and counting of cells were as described (4, 8, 11). For experiments, cells were grown in surface culture in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) calf serum. Subconfluent monolayers were detached by trypsin, washed by centrifugation with Hanks' balanced salt solution (HBSS), and plated in 6or 24-well dishes (GIBCO) in DMEM/10%6 calf serum at a density of 8 x 103 cells/cm2. After 4 hr of incubation at 370C in a humidified incubator gassed with 5% C02/95% air, the medium was aspirated; the monolayers were washed with HBSS; and serum-free medium (4), without insulin (or IGFI), and the indicated concentrations of Met-hGH (or other hormones being tested) were added. Colony-forming efficiency was determined as described (4). Experiments were run in triplicate for each time point, and each point was plated out in at least duplicate. The plating efficiency was generally 50% and 25% for cells in DMEM/ 10% calf serum and serum-free medium (without GH or insulin), respectively. Differentiation was quantified by determining the specific activity of the adipose marker enzyme GPDH as described (4). Cellular commitment times for adipose differentiation in response to GH were determined in serum-free medium. Cells were grown to near confluence in the nonadipogenic medium of DMEM/5% cat serum. Monolayers were then washed extensively with HBSS followed by the addition of serum-free medium as described (4). The zero time of the experiment was at the addition of serum-free medium. At this time the serum-free medium contained Met-hGH (2 nM) and insulin (1.6 MLM), a condition that induces adipose differentiation (4). After the indicated times (see Fig. 1C), the medium was aspirated, and the monolayers were washed extensively with HBSS. After washing, the residual levels of Met-hGH were determined by radioimmunoassay and were '1.0 x 10-13 M, which is two or three orders of magnitude lower than the EC50 for Met-hGH-induced adipose differentiation of 3T3-F442A preadipocytes (4, 6). After washing, the cells were fed fresh serum-free medium without GH and incubated for 20 days. Then the extent of differentiation was determined by assaying GPDH-specific activities. The positive control for the commitment experiment was cells maintained in the original serum-free medium with Met-hGH (2 nM) and insulin (1.6 ,uM) for the entire 20 days (i.e., full differentiation as a function of time of GH exposure). Each point is the average of triplicate determinations. DNA synthesis (7) was assayed 18-24 hr after the addition of FBS or PDGF plus insulin to cultures by aspirating the medium and replacing it with identical medium containing 1-10 ,uCi of [3H]thymidine per ml. After a 1-hr pulse, the isotope was removed, the monolayers were washed with HBSS, and DNA was precipitat |
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| Language | English |
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