No abstract

The bone marrow stroma contains pre-adipocyte cells which are part of the hemopoietic microenvironment. Cloned stromal cell lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine stromal cell line and compared to other stromal and pre-adipocyte cell lines. In long-term cultures, the +/+2.4 stromal cells support myeloid cell growth, consistent with their expression of macrophage-colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 cells failed to support stromal cell dependent B lineage lymphoid cells in vitro, suggesting that these stromal cells exhibit only a myelopoietic support function. The +/+2.4 cells differentiate into adipocytes spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of agonists based on morphologic and gene marker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination, a time dependent increase in the steady state mRNA and enzyme activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis was accompanied by a slight decrease in the signal intensity of the macrophage-colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow stromal cell lines. These data demonstrate that although the lympho-hematopoietic support function of pre-adipocyte bone marrow stromal cell lines is heterogeneous, they share a common mechanism of adipogenesis.

Antioxidants are protective against septic shock in animal models. Recently, free radical scavengers have been found to inhibit the activation of the NF-KB protein in a number of cell lines. This transcriptional regulatory protein binds to the promoters of the proinflammatory cytokines tumor necrosis factor, interleukin-6, and the macrophage inflammatory proteins. The current work examined lipopolysaccharide-induced NF-KB activation in the J774 macrophage-like cell line and primary peritoneal macrophages from lipopolysaccharide-responsive (C3HeB/Fej) and -nonresponsive (C3H/HeJ) murine strains. The DNA-binding activity of the NF-KB protein directly correlated with mRNA expression for the genes encoding the proinflammatory cytokines and the free radical scavenging enzyme, superoxide dismutase. Both the p50 and p65 NF-KB subunits were detected on gel supershift assays. Minimal NF-KB activity was observed following exposure of C3H/HeJ macrophages to lipopolysaccharide. The antioxidant dimethyl sulfoxide decreased the level of NF-KB activation in the J774 cells. This correlated with decreased expression of cytokine mRNAs and tumor necrosis factor bioactivity. These results suggest that modulation of NF-KB activation may provide a mechanism through which antioxidants protect against endotoxemia in murine models.

The 52-kDa phosphoprotein, also reported as lymphocyte-specific gene 1 and WP34, is transcribed as a 1.6-kb mRNA in B lymphocytes, B cell lines, and untransformed T cells. This gene encodes a cytoplasmic and plasma membrane-associated protein that is phosphorylated at a casein kinase II site and reportedly binds calcium. Based on these properties, it has been hypothesized that lymphoid form of the 52-kDa phosphoprotein protein may play a role in lymphocyte signal transduction. We show that alternatively spliced mRNA are expressed from this gene in nonlymphoid cell lines (myocytes, stromal cells, fibroblasts). These cell lines do not express the 1.6-kb lymphoid cell-specific transcript. Instead, mRNA of 2.0 and 2.8 kb are detected in varying abundance. A full-length 2.0-kb cDNA has been cloned and sequenced from the BMS2 stromal cell line by conventional screening and polymerase chain reaction-based methods. This cDNA clone, designated S37, has a single open reading frame encoding a 328 amino acid peptide. The nucleotide sequence of the S37 stromal cell cDNA is identical to that of the lymphocyte derived pp52 cDNA from the 3' poly(A) tail to the codon encoding the amino acid at residue 24. This region of the S37 cDNA clone encodes a protein that is identical to that encoded by the lymphoid pp52 cDNA and includes a casein kinase II phosphorylation site. However, the two clones differ in their 5' nucleotide sequence and their NH3 terminal amino acid sequence. This organization is consistent with alternative exon utilization. These results suggest that tissue-specific control mechanisms are used to generate different forms of lymphoid form of the 52-kDa phosphoprotein mRNA in lymphoid cells versus mesoderm-derived, nonlymphoid cell lineages.