A series of mutations in the D-MEF2 transcription factor reveal multiple functions in larval and adult myogenesis in Drosophila.

The D-mef2 gene encodes a MADS domain transcription factor expressed in differentiated muscles and their precursors in the Drosophila embryo. Embryos deficient for D-MEF2 protein due to a deletion of upstream transcriptional control sequences fail to form muscle, suggesting that the gene is required for muscle cell differentiation. To directly demonstrate a role for D-mef2 in embryonic myogenesis, we isolated gene mutants containing EMS-induced point mutations, characterized the effects of these mutations on D-MEF2 protein stability and nuclear localization, and analyzed the resulting muscle phenotypes. Our results show that in the somatic muscle lineage, D-mef2 is required for both the formation and patterning of body wall muscle. In the absence of somatic myogenesis, there is extensive apoptosis among the myoblast cell population. In contrast, in the cardiac muscle lineage, morphogenesis of the dorsal vessel occurs normally but the three myosin subunit genes are not expressed. Mutant embryos also exhibit an abnormal midgut morphology, which correlates with the absence of alpha PS2 integrin gene expression and muscle-specific enhancer function, suggesting that D-mef2 regulates the inflated locus which encodes this integrin subunit. D-MEF2 is also expressed in adepithelial cells and rare D-mef2 transheterozygous mutant adults fail to fly, consistent with defects observed in the indirect flight muscles. These results demonstrate that the D-mef2 gene has multiple functions in myogenesis and tissue morphogenesis during Drosophila development.

Changes in sulfated proteoglycan production after activation of rat liver macrophages.

Production of extracellular matrix proteins-in particular, the proteoglycans-by macrophages is important in many of their functions, including cell-cell recognition, adhesion and phagocytosis. In this study, we characterized changes in sulfated proteoglycan production by hepatic macrophages following in vivo activation with lipopolysaccharide. We found that both resident Kupffer cells and liver macrophages from lipopolysaccharide-treated rats incorporated [35S]sulfate into proteoglycans. Lipopolysaccharide-activated macrophages incorporated two to three times more of the label than did resident Kupffer cells. In addition, although both cell types produced chondroitin sulfate and heparan sulfate, resident Kupffer cells synthesized more chondroitin sulfate whereas lipopolysaccharide-activated cells produced more heparan sulfate. Using specific antibodies and flow cytometry, we also found that hepatic macrophages produced chondroitin-4-sulfate, chondroitin-6-sulfate and chondroitin-O-sulfate. Lipopolysaccharide-activated macrophages contained more chondroitin-4-sulfate and chondroitin-O-sulfate and less heparan sulfate than did resident Kupffer cells. Both tunicamycin and beta-D-xylosides, inhibitors of sulfated proteoglycan biosynthesis, were found to block phagocytosis by the cells. Taken together, these results suggest that sulfated proteoglycans are important in activation and functional responsiveness of liver macrophages.

Alterations in epidermal sulfated proteoglycan production following topical application of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate to mouse skin.

Topical application of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin causes marked changes in epidermal cell growth and differentiation. In the present studies we characterized the production of sulfated proteoglycans in the epidermis following treatment with TPA since these macromolecules are important structural and functional components of the tissue. We found that 35S-sulfate was readily incorporated into mouse epidermal proteoglycans. Sepharose CL-4B column chromatography revealed one major peak of sulfated proteoglycans in this tissue (Kav = 0.4-0.5). Approximately 65% of these proteoglycans were heparan sulfate and 10-20% chondroitin sulfate. Using specific monoclonal antibodies and flow cytometry, we found that the epidermal cells produced chondroitin-4-sulfate, chondroitin-6-sulfate and chondroitin-O-sulfate. Within 24 hr of application of TPA to mice, an increase in glycosaminoglycan content of the epidermis was observed. This was associated with a decrease in 35S-sulfate uptake into the tissue. Although TPA had no effect on the size or relative distribution of the epidermal sulfated proteoglycans, an increase in chondroitin-4-sulfate expression was observed in treated skin. Changes in the production of proteoglycans following TPA treatment may underlie structural alterations that occur in the epidermis during tumor promotion.

Distinct patterns of sulfated proteoglycan biosynthesis in human monocytes, granulocytes and myeloid leukemic cells.

The production of sulfated proteoglycans was compared in mature peripheral blood granulocytes and monocytes and HL-60 promyelocytic leukemia cells. We found that HL-60 cells synthesized 5-10 times more proteoglycans than peripheral blood leukocytes. Differentiation of HL-60 cells toward mature monocytes by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or towards granulocytes by treatment with retinoic acid or dibutyryl cyclic adenosine-3',5'-monophosphate (dbcAMP) resulted in a small (20-30%) decrease in sulfated proteoglycan biosynthesis. Chondroitin sulfate was found to be the predominant proteoglycan produced by monocytes, PMN and undifferentiated HL-60 cells. Differentiated HL-60 cells produced chondroitin sulfate as well as sulfated proteoglycans sensitive to nitrous acid degradation. Similar results were observed in TPA, dbcAMP and retinoic acid differentiated HL-60 cells indicating that the changes in proteoglycan biosynthesis observed were independent of the developmental pathway. Using specific monoclonal antibodies and flow cytometry, we also found that HL-60 cells and monocytes produced chondroitin-4-sulfate, chondroitin-6-sulfate and chondroitin-O-sulfate while PMN only produced chondroitin-4-sulfate. In addition, although there were no significant differences in antibody binding to undifferentiated and differentiated HL-60 cells, the tumor cells bound 5-20 times more of the antibodies than the peripheral blood leukocytes. Our data demonstrate that sulfated proteoglycan production by HL-60 cells is distinct from PMN and monocytes. In addition, the fact that differentiated HL-60 cells continue to synthesize larger amounts of the proteoglycans than the peripheral blood leukocytes indicates these cells have not completely matured.