The goal of these investigations is to understand the regulation of cytoskeletal biosynthesis and function during mammalian cell development. The system utilized is the differentiation of mouse 3T3-adipocytes, where large and specific decreases in mRNA levels for actin and tubulin appear to play an important regulatory role in the differentiation process. The proposed experiments, which emphasize the regulation of cytoskeletal gene expression, cell structure and lipogenesis, are particularly relevant to human diseases involving fat cell number and function, such as obesity, diabetes and liposarcoma. Experiments underway use cloned cDNA probes for Beta-actin and Beta-tubulin to assay transcription during differentiation in both isolated nuclei and whole cells. If transcriptional changes cannot quantitatively account for changes in these mRNAs, we will study mRNA turnover and processing. The build-up and processing of nuclear RNAs will get particular attention in light of evidence suggesting the build-up of a putative actin mRNA precursor during differentiation. The actin gene active in adipocytes will be isolated and used to define the primary transcription unit and to ask if this putative actin precursor has a structure consistent with such a role. Further studies involve construction of in vitro systems to study relevant control mechanisms which are operating. Experiments will be performed to understand the normal physiological role of the cytoskeletal and morphological regulation of lipogenic gene expression shown to exist in adipocytes. In particular, the role of cyclic AMP's effects on cell adhesion and the cytoskeleton in the suppression of lipogenic gene expression by this key physiological agent will be examined by exposing differentiating cells to a variety of cyclic AMP agents while quantitatively varying substrate adhesiveness or treating with anticytoskeletal drugs. The subsequent expression of lipogenic protein and RNA will be studied with antibodies and cDNA clones previously constructed. Finally, the reversibility of cellular and molecular differentiation-dependent changes will be studied. This is now experimentally approachable by replating differentiated cells which have had lipid accumulation blocked and hence, retain firm attachment to the sub-stratum. Subsequent cell growth and cytoskeleton synthesis will be studied at the protein and RNA levels.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Dana-Farber Cancer Institute
United States
Zip Code
Rosen, B S; Cook, K S; Yaglom, J et al. (1989) Adipsin and complement factor D activity: an immune-related defect in obesity. Science 244:1483-7
Kitagawa, K; Rosen, B S; Spiegelman, B M et al. (1989) Insulin stimulates the acute release of adipsin from 3T3-L1 adipocytes. Biochim Biophys Acta 1014:83-9
Rauscher 3rd, F J; Sambucetti, L C; Curran, T et al. (1988) Common DNA binding site for Fos protein complexes and transcription factor AP-1. Cell 52:471-80
Rosoff, P M; Burakoff, S J; Greenstein, J L (1987) The role of the L3T4 molecule in mitogen and antigen-activated signal transduction. Cell 49:845-53
Flier, J S; Cook, K S; Usher, P et al. (1987) Severely impaired adipsin expression in genetic and acquired obesity. Science 237:405-8
Distel, R J; Ro, H S; Rosen, B S et al. (1987) Nucleoprotein complexes that regulate gene expression in adipocyte differentiation: direct participation of c-fos. Cell 49:835-44
Cook, K S; Min, H Y; Johnson, D et al. (1987) Adipsin: a circulating serine protease homolog secreted by adipose tissue and sciatic nerve. Science 237:402-5