Latent transforming growth factors beta (TGF-β) are easily detectable in embryonic and adult hematopoietic tissues and in vitro studies show that they are potent antagonists of lymphopoiesis and ...myelopoiesis when converted to biologically active form. To learn more about possible roles in hematopoiesis, active TGF- β1, was added to cultures prepared to support myeloid cells (Dexter conditions) or B lineage lymphocytes (Whitlock-Witte conditions) and studied in detail. Hematopoiesis was permanently arrested in Dexter cultures treated with 40 pmol/L (1 ng/mL) of active TGF-β from initiation. In addition, adipogenesis was inhibited in a dose-dependent manner, and adherent layers from treated cultures were defective when recharged with fresh bone marrow cells. Ongoing neutrophil production was terminated in established cultures when addition of the factor was delayed for 8 weeks. In contrast, in experiments with Whitlock-Witte cultures, some of the flasks produced lymphocytes in the continuous presence of TGF- β1, (40 pmol/L). Lymphopoiesis was completely arrested by tenfold higher concentrations, and this was most effective when added at the beginning of culture. Precursors of lymphocytes as well as the microenvironmental elements necessary for supporting their growth survived 2 weeks of cytokine treatment (400 pmol/L) in Dexter cultures. Normal outgrowth of lymphocytes occurred when the cultures were switched to Whitlock-Witte conditions. Surface marker expression on lymphocytes growing in TGF-β1 resistant or previously treated cultures was not unusual. These studies demonstrate that TGF- β is a negative regulator of hematopoiesis in long-term cultures and show that this includes effects on microenvironmental elements. At low concentrations, production of myeloid cells was preferentially affected.
OBJECTIVE: Genes encoding the circadian transcriptional apparatus exhibit robust oscillatory expression in murine adipose tissues. This study tests the hypothesis that human subcutaneous ...adipose-derived stem cells (ASCs) provide an in vitro model in which to monitor the activity of the core circadian transcriptional apparatus. RESEARCH METHODS AND PROCEDURES: Primary cultures of undifferentiated or adipocyte-differentiated ASCs were treated with dexamethasone, rosiglitazone, or 30% fetal bovine serum. The response of undifferentiated ASCs to dexamethasone was further evaluated in the presence of lithium chloride. Lithium inhibits glycogen synthase kinase 3, a key component of the circadian apparatus. Total RNA was harvested at 4-hour intervals over 48 hours and examined by real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Adipocyte-differentiated cells responded more rapidly to treatments than their donor-matched undifferentiated controls; however, the period of the circadian gene oscillation was longer in the adipocyte-differentiated cells. Dexamethasone generated circadian gene expression patterns with mean periods of 25.4 and 26.7 hours in undifferentiated and adipocyte-differentiated ASCs, respectively. Both rosiglitazone and serum shock generated a significantly longer period in adipocyte-differentiated ASCs relative to undifferentiated ASCs. The Bmal1 profile was phase-shifted by ~8 to 12 hours relative to Per1, Per3, and Cry2, consistent with their expression in vivo. Lithium chloride inhibited adipogenesis and significantly lengthened the period of Per3 and Rev-erbα gene expression profiles by >5 hours in dexamethasone-activated undifferentiated ASCs. DISCUSSION: These results support the initial hypothesis and validate ASCs as an in vitro model for the analysis of circadian biology in human adipose tissue.
The murine immunoglobulin κ gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots ...of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific ~0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine κ enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5′ end of the nuclease-susceptible region.
The effects of soluble mediators and medium supplements commonly used to induce chondrogenic differentiation in different cell culture systems were investigated to define their dose-response profiles ...and potentially synergistic effects on the chondrogenic differentiation of adipose-derived adult stromal (ADAS) cells. Human ADAS cells were suspended within alginate beads and cultured in basal medium with insulin, transferrin, and selenious acid (ITS+) or fetal bovine serum (FBS) and treated with different doses and combinations of TGF-beta1 (0, 1, and 10 ng/mL) and dexamethasone (0, 10, and 100 nM). Cell growth and chondrogenic differentiation were assessed by measuring DNA content, protein and proteoglycan synthesis rates, and proteoglycan accumulation. The combination of ITS+ and TGF-beta1 significantly increased cell proliferation. Protein synthesis rates were increased by TGF-beta1 and dexamethasone in the presence of ITS+ or FBS. While TGF-beta1 significantly increased proteoglycan synthesis and accumulation by 1.5- to 2-fold in the presence of FBS, such effects were suppressed by dexamethasone. In summary, the combination of TGF-beta1 and ITS+ stimulated cell growth and synthesis of proteins and proteoglycans by human ADAS cells. The addition of dexamethasone appeared to amplify protein synthesis but had suppressive effects on proteoglycan synthesis and accumulation.
Recent advances in long-term bone marrow (BM) culture techniques have allowed investigators to dissect cellular components responsible for lympho hematopoiesis. Consequently, a number of "stromal" ...cell clones have been developed which are capable of supporting B lineage lymphocyte growth and proliferation in vitro by direct cell-cell interactions and the release of cytokines. While much work has focused on the support function of these cells, questions remain regarding their own differentiation potential. We have examined adipogenesis in the cloned BM stromal cell, BMS2. The presence of hydrocortisone, methylisobutylxanthine, or 30% fetal calf serum each accelerated adipocyte differentiation. This process was accompanied by the accumulation of triglycerides and cholesterol esters along with the induction of adipocyte-specific enzymes. Likewise, the steady-state level of mRNA transcripts increased for genes related to lipid metabolism. However, the pattern of mRNA expression in BMS2 adipocytes differed from that of a well-established, pre-adipocyte cell line, 3T3-L1, with respect to the following genes: glycerol phosphate dehydrogenase, CAAT/enhancer binding protein and angiotensinogen. Adipocyte BMS2 cells retailed the ability to support stromal cell-dependent B lineage lymphocytes in methylcellulose assays. The adipocytes continued to express macrophage-colony-stimulating factor mRNA constitutively and interleukin 6 mRNA in an inducible manner, similar to the BMS2 pre-adipocytes. Together, these data document a close developmental relationship between a specialized fibroblasts and adipocytes in the BM and suggest that adipocyte stromal cells may play an active role in lympho-hematopoiesis.
Proteins capable of interacting with the enhancer of the immunoglobulin kappa gene in vitro have been detected in extracts of nuclei from human B cells and from human, mouse, and rabbit spleens. The ...experiments, based on an exonuclease protection technique, demonstrate nuclear protein factors binding to a 30- to 35-base-pair domain containing both the simian virus 40 enhancer core element (TTTCCA) and the octamer CAGGTGGC that was previously identified as the consensus sequence for protein-binding sites in the murine immunoglobulin heavy-chain enhancer. This 30- to 35-base-pair domain in the human kappa enhancer is homologous to a site of protein binding detected in the murine kappa enhancer by other investigators using a gel retardation assay. Our results complement in vivo dimethyl sulfate footprinting studies of the human immunoglobulin kappa enhancer which demonstrated B cell-specific changes in guanine reactivity immediately 5′ to the consensus octamer. Together, these findings suggest that DNA-binding proteins in B-cell nuclei interact with the 5′ portion of the human kappa-gene enhancer. Such proteins could play a role in the B cell-specific transcription of the human immunoglobulin kappa gene.
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