Elena J. Moerman

Aging activates adipogenic and suppresses osteogenic programs in mesenchymal marrow stroma/stem cells: the role of PPAR-γ2 transcription factor and TGF-β/BMP signaling pathways

Osteoblasts and adipocytes originate from a common progenitor, which arises from bone marrow mesenchymal stroma/stem cells (mMSC). Aging causes a decrease in the number of bone‐forming osteoblasts and an increase in the number of marrow adipocytes. Here, we demonstrate that, during aging, the status of mMSC changes with respect to both their intrinsic differentiation potential and production of signaling molecules, which contributes to the formation of a specific marrow microenvironment necessary for maintenance of bone homeostasis. Aging causes a decrease in the commitment of mMSC to the osteoblast lineage and an increase in the commitment to the adipocyte lineage. This is reflected by changes in the expression of phenotype‐specific gene markers. The expression of osteoblast‐specific transcription factors, Runx2 and Dlx5, and osteoblast markers, collagen and osteocalcin, is decreased in aged mMSC. Conversely, the expression of adipocyte‐specific transcription factor PPAR‐γ2, shown previously to regulate osteoblast development and bone formation negatively and to regulate marrow adipocyte differentiation positively, is increased, as is a gene marker of adipocyte phenotype, fatty acid binding protein aP2. Furthermore, production of an endogeneous PPAR‐γ activator(s) that stimulates adipocyte differentiation and production of autocrine/paracrine factor(s) that suppresses the osteoblastic phenotype are also increased. In addition, expression of different components of TGF‐β and BMP2/4 signaling pathways is altered, suggesting that activities of these two cytokines essential for bone homeostasis change with aging.

Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPARγ2

Cells of the bone marrow stroma can reversibly convert among different phenotypes. Based on this and on evidence for a reciprocal relationship between osteoblastogenesis and adipogenesis, we have isolated several murine bone marrow‐derived clonal cell lines with phenotypic characteristics of osteoblasts or adipocytes, or both. Consistent with a state of plasticity, cell lines with a mixed phenotype synthesized osteoblast markers like type I collagen, alkaline phosphatase, osteocalcin, as well as the adipocyte marker lipoprotein lipase, under basal conditions. In the presence of ascorbic acid and β‐glycerophosphate—agents that promote osteoblast differentiation—they formed a mineralized matrix. In the presence of isobutylmethylxanthine, hydrocortisone, and indomethacin—agents that promote adipocyte differentiation—they accumulated fat droplets, but failed to express adipsin and aP2, markers of terminally differentiated adipocytes. Furthermore, they were converted back to matrix mineralizing cells when the adipogenic stimuli were replaced with the osteoblastogenic ones. A prototypic cell line with mixed phenotype (UAMS‐33) expressed Osf2/Cbfa1—a transcription factor required for osteoblast differentiation, but not PPARγ2—a transcription factor required for terminal adipocyte differentiation. Stable transfection with a PPARγ2 expression construct and activation with the thiazolidinedione BRL49653 stimulated aP2 and adipsin synthesis and fat accumulation, and simultaneously suppressed Osf2/Cbfa1, α1(I) procollagen, and osteocalcin synthesis. Moreover, it rendered the cells incapable of forming a mineralized matrix. These results strongly suggest that PPARγ2 negatively regulates stromal cell plasticity by suppressing Osf2/Cbfa1 and osteoblast‐like biosynthetic activity, while promoting terminal differentiation to adipocytes. J. Cell. Biochem. 74:357–371, 1999.

Divergent Effects of Selective Peroxisome Proliferator-Activated Receptor-γ2 Ligands on Adipocyte Versus Osteoblast Differentiation

PPARγ is activated by diverse ligands and regulates the differentiation of many cell types. Based on evidence that activation of PPARγ2 by rosiglitazone stimulates adipogenesis and inhibits osteoblastogenesis in U-33/γ2 cells, a model mesenchymal progenitor of adipocytes and osteoblasts, we postulated that the increase in marrow fat and the decrease in osteoblast number that occur during aging are due to increased PPARγ2 activation. Here, we show that the naturally occurring PPARγ ligands 9,10-dihydroxyoctadecenoic acid, and 15-deoxy-Δ12,14-PGJ2, also stimulate adipocytes and inhibit osteoblast differentiation of U-33/γ2 cells. Strikingly, 9,10-epoxyoctadecenoic acid and the thiazolidine acetamide ligand GW0072 [(±)-(2S,5S)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thaizolidineN,N-dibenzyl-acetamide] prevent osteoblast differentiation, but do not stimulate adipogenesis, whereas 9-hydroxyoctadecadienoic acid stimulates adipogenesis but does not affect osteoblast differentiation. The divergent effects o...

Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence.

Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid fibroblasts (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing 20% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interpersed nuclear element I (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of diverse genes which may play a role in the senescent arrest of cellular replication and in the genesis of WS, normal biological aging, and attendant diseases.

Heat-Labile Enzymes in Skin Fibroblasts from Subjects with Progeria

To characterize further the genetic basis of progeria, thermolability studies were performed on three genetically distinct enzymes in crude extracts of cultured skin fibroblasts derived from two subjects with that syndrome. At early passage the progeric fibroblasts, as compared to controls, contained a significantly higher percentage of heat-labile glucose-6-phosphate dehydrogenase (12.83 plus or minus 1.72 vs 1.11 plus or minus 0.44 [mean plus or minus S.E.M.], p smaller than 0.001), 6-phosphogluconate dehydrogenase (9.71 plus or minus 0.68 vs. 0.67 plus or minus 0.22, p smaller than 0.001), and hypoxanthine-guanine phosphoribosyltransferase (31.41 plus or minus 1.89 vs 7.67 plus or minus 1.71, p smaller than 0.001), and the differences were maintained throughout the in vitro life-span. These data, in conjunction with previous reports of defective HL-A antigens, indicate a widespread defect in genetic expression. The most likely cause appears to be an aberration in protein synthesis or degradation, or both, although multiple somatic mutations cannot be ruled out. Increased thermolability of enzymes in cultured cells may provide a screening test for persons predisposed to progeria and other disorders of premature aging.

Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts.

Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts. Molar ratios of IGFBP-3/IGF-II were always higher in senescent cultures and maintained a hierarchy of old > WS > young human diploid fibroblasts. Transfection into fibroblasts of the normal full-length IGFBP-3 cDNA in an expression vector resulted in a significant reduction in colony formation compared to cells transfected with an empty expression vector (no cDNA) or with IGFBP-3 cDNA altered by a 273 base pair (bp) deletion. Addition to old and young cultures of recombinant human IGFBP-3 and IGF-I at 1:1 or 5:1 molar ratios inhibited IGF-I-mediated DNA synthesis by approximately 70-80%. These data indicate that IGFBP-3 may play an important role in the quiescent and senescent growth arrest of HDF.

Studies on the molecular-genetic basis of replicative senescence in Werner syndrome and normal fibroblasts

Based on evidence that human diploid fibroblasts (HDF) from the Werner syndrome (WS) of premature aging might overexpress an inhibitor of DNA synthesis (IDS), we prepared a eukaryotic cDNA expression library from WS mRNA and tested it for IDS activity in a transient assay. Two of six WS cDNA pools tested gave IDS activity, then on plus/minus screening revealed several differentially expressed cDNA clones. By slot blot and Northern analysis, one cDNA clone was found to be overexpressed in WS and normal senescent HDF, but not in quiescent normal HDF, indicating that it is senescence-specific. Further studies are needed to clarify: a) whether this cDNA truly acts as an IDS; b) if so, whether it acts alone or in concert with other cDNAs; and c) whether it is involved in the degenerative and malignant sequelae of WS and normal aging.

Characterization Of IGFBP-3, PAI-1 and SPARC mRNA expression in senescent fibroblasts

The RNA species encoded by IGFBP-3 (insulin-like growth factor binding protein-3), PAI-1 (plasminogen activator inhibitor-1) and SPARC (secreted protein-acidic and rich in cysteine; a.k.a. osteonectin) are overexpressed in senescent human diploid fibroblasts (HDF). Their extracellular products have the ability to modulate cell growth in culture and have been shown to have inhibitory effects on DNA synthesis and/or cell growth. This overproduction may contribute to a number of features of aging, including osteoporosis, atherosclerosis and diabetes mellitus type II. Based on analysis of steady-state mRNA levels, which showed similar patterns for all three along with overexpression in senescent cells, we further investigated their transcription rates and stability to determine reasons for their overexpression and to determine if coordinate gene regulation was involved. Characterization of the rates of transcription and the levels of message stability of these genes in early passage (young) versus late passage (old) HDF revealed that IGFBP-3, PAI-1 and SPARC are coordinately overexpressed but not regulated by a unique or simple mechanism encompassing all three transcripts. Only PAI-1 shows an increase in the rate of transcription, while all three show evidence that their overexpression is due to an increase in the stability of RNA. Thus, the overexpression of these genes in senescent fibroblasts involves interactions not only at the transcriptional level but also with protein factors involved in determining the stability and the degradation of RNA.

Expression of senescence-induced protein WS3-10 in vivo and in vitro

In our efforts to characterize cellular senescence we have shown that the mRNA encoding WS3-10 protein is overexpressed in senescent human diploid fibroblasts (HDF) when compared with their younger counterparts, and that forced expression of the WS3-10 cDNA in young HDF results in suppression of calcium-dependent membrane currents, presumably due in part to the presence of a calcium binding domain within the WS3-10 protein. We have now expressed this protein in E. coli and have obtained affinity purified antibodies. Western blot analysis utilizing these antibodies showed that WS3-10 protein is also overexpressed in senescent HDF when compared to young HDF, and in normal fetal lung HDF when compared to SV40-transformed fetal lung HDF. HeLa cells do not express WS3-10 protein. In addition, we looked for WS3-10-related species in a variety of rat tissues. Analysis of WS3-10 immunologically related proteins in rat tissue extracts revealed two WS3-10 homologs, sized 22 kDa and 20 kDa. The latter presumably result from proteolytic removal of the C-terminal end of the 22 kDa polypeptide. The ratio between these polypeptides varies in a tissue-specific manner. Two proteins immunologically related to WS3-10 with sizes of 39 kDa and 91 kDa were present in rat spleen and skeletal muscle, respectively.