Abstract
We have identified 41 novel and many previously known growth response genes induced in regenerating liver and insulin-treated Reuber H35 cells, a rat hepatoma cell line that grows in response to physiologic concentrations of insulin and retains some properties of regenerating liver. Although many genes are expressed similarly in the two systems, there are important differences in the kinetics of induction of some genes. These differences allowed us to identify and characterize novel genes that are highly insulin-induced and expressed as delayed-early genes in regenerating liver. Sequence analysis of CL-6, the most abundant insulin-induced gene, resulted in the identification of a highly hydrophobic hepatic protein. Sequence analysis of HRS, a highly insulin-induced delayed-early gene, demonstrated that it is a member of the family of regulators of alternative pre-mRNA splicing. Different forms of HRS mRNA are temporally regulated during the growth response, suggesting that HRS could autoregulate processing of its pre-mRNA. Given the dramatic increase in RNA production during late G1, proteins induced by mitogens like insulin that control RNA processing are likely to have important roles in cell cycle regulation.
Highlights
We have identified 41 novelandmanypreviously circulating hormones, growth factors, and known growth responsgeenes induced in regenerating nervous input participate in the regulation of this response, liver and insulin-treatedReuber H35 cells, a rat hep- but the actual mechanism remainsincompletely understood
Other studiessuggest that insulinmay quence analysis of HRS,a highly insulin-induced de- be important in threegulation of rRNA and protein synthesis layed-early gene, demonstrated that itis a member of (7-9) which occur later in G1
Studies of effects of insulin on thefamilyofregulatorsof alternative pre-mRNA regeneration have been difficult because hepatocytes in culsplicing.Differentforms oHf RS mRNA aretemporally ture do not demonstrate normal growth regulation, and in regulated during the growth responsue,ggestingthat intact animals, there is a lack of measurable parameters of HRS could autoregulate processingof its pre-mRNA. growth control other than the assessmenotf DNA synthesis
Summary
Cell Culture-H-35 cells weregrown in low glucoseDulbecco’s modified Eagle’s medium, low glucose (DMEM,’ Life Technologies, Inc.) supplemented with 5% fetal bovine serum (FBS, Life Technologies, Inc.), 5% calf serum (Life Technologies, Inc.), 2 mM L-glutamine (Gln, Flow) and 100 units of penicillin and 50 units/ml streptomycin (Flow) as previously reported (23). The medium was changed to serum-free DMEM for 72 h, at which time the cells were between 50 and 80% confluent. Several milligrams of purified protein were obtained and sent toCocalico Biologicals(Reamstown, PA) for injection into rabbits for polyclonal antisera. Immunoblots were made from either purified PET-CL-6 fusion protein (100 ng/lane), or cell extracts from 3T3, H35, or liver (30) cells after resuspending whole cells in SDS loading buffer, boiling for 15 min, and electrophoresing on 12-15% SDS-polyacrylamide gels. Immunoblots were incubated with eithera 1:lOOO preimmune serum or 1:lOOO anti-PET-CL-6 antisera. Balb/c 3T3 cells weregrown in DMEM supplemented with 10% FBS, Gln, and streptomycin.
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