(Received for publication, February 12, 1996; and in revised form, March 7, 1996)
From the
In mouse preadipocyte Ob1771 cells, transcription of the insulin-like growth factor-I (IGF-I) gene was stimulated by growth hormone (GH), and IGF-I protein combined with GH in medium was required for their differentiation to adipocytes. During induction of the differentiation, the intracellular expression of each class of IGF-I mRNA was analyzed by reverse transcriptase-polymerase chain reaction. When the cells were cultured in the presence of GH, the class 1del. IGF-I mRNA was a major molecular species among IGF-I mRNAs. In the presence of both GH and IGF-I, the splicing pattern of IGF-I mRNA changed from class 1del. to class 1. Moreover, as detected by Western blotting, the IGF-I protein was present in cells and in the medium only when the cells were cultured in the presence of both GH and IGF-I. We found that IGF-I secreted from Ob1771 cells could act in an autocrine/paracrine fashion and induce the differentiation of other Ob1771 cells. It was demonstrated that the translation efficiency of class 1 mRNA was higher than that of class 1del. mRNA in vitro. These results suggested that stimulation with exogenous IGF-I in the presence of GH was required for the production of class 1 IGF-I mRNA and that the production of the IGF-I protein was activated by increasing the translation efficiency through shifting the splicing pattern of IGF-I mRNA from class 1del. to class 1. Exogenous IGF-I triggered the differentiation by initiating the synthesis of endogenous IGF-I.
Insulin-like growth factor-I (IGF-I) ()is a 70-amino
acid polypeptide similar to proinsulin(1) . Transcription of
the IGF-I gene is regulated by growth hormone (GH), and IGF-I is
thought to mediate many of the biological effects of
GH(2, 3, 4, 5, 6) . IGF-I
has insulin-like activities such as stimulation of glycogen synthesis (7) . IGF-I also functions as a mitogen and as a
differentiation factor for various cell lines, including
preadipocytes(8) . The biological actions of IGF-I begin by
interaction with its cell surface receptor, which is a ligand-activable
tyrosine-specific protein kinase similar to the insulin
receptor(9) .
In mouse and rat(10, 11) , the IGF-I genes have two leader exons (exons 1 and 2), resulting in two kinds of mRNAs (classes 1 and 2) (11, 12, 13) . There is another mRNA species, class 1del., in which a central region of exon 1 is missing (Fig. 1A)(14, 15) . Exon 5 is also spliced alternatively, resulting in Ea encoded by exons 4 and 6 and Eb encoded by exons 4, 5, and 6(10, 16) . These diverse IGF-I mRNAs eventually give the same mature protein. The biological significance of the diversity of mRNAs, signal peptides, and E domains is not understood.
Figure 1: Structure of mouse IGF-I gene and expression of each class of IGF-I mRNAs during induction of differentiation. A, structure of mouse IGF-I gene is shown. Boxes indicate exons, and lines indicate introns and flanking regions. Solid boxes mark the coding region for the IGF-I prepropeptide, and a hatched box is the region spliced out, giving class 1del. mRNA. Half-arrows above the exons indicate positions and orientations of the primers used for RT-PCR. Three classes of mature mRNAs are also shown at the bottom of the panel. Ea and Eb are the splicing variants resulting from alternative splicing of exon 5. B, Ob1771 cells were grown to confluence in the standard medium (day 0) and cultured up to day 4 in the GH differentiation medium or in the GH-IGF-I differentiation medium. The medium was changed to the standard medium and the cells were cultured for 2 days. On the day indicated on top of the panel, total RNA was prepared from the cells. Using 1 µg of RNA, RT-PCR was done with primers 1 and 6 (class 1), with primers 1-1del. and 6 (class 1 + class 1del.), and with primers 2 and 6 (class 2). Amplified DNA fragments were analyzed by Southern hybridization as described under ``Experimental Procedures.'' Similar results were obtained in three independent experiments.
Mouse preadipocyte Ob1771 cells (17) can differentiate to adipocytes. GH has a strong adipogenic activity in Ob1771, 3T3-F442A(18, 19, 20) , and 3T3-L1 (21) preadipocytes. In Ob1771 cells, GH stimulates the formation of diacylglycerol(22) , modulates the transcription of the lipoprotein lipase gene (23) and transiently increase the transcription of the c-fos gene(22) . GH also stimulates the transcription of the IGF-I gene(6) , and IGF-I is thought to participate in inducing the differentiation. In differentiated Ob1771 cells, enzymes for lipid synthesis, such as glycerophosphate dehydrogenase (GPDH), are activated(24) .
Transcription of the IGF-I gene is stimulated by GH, but we observed that IGF-I combined with GH was essential for the differentiation of Ob1771 cells. The following interpretations are possible for these facts. (i) For some reason, IGF-I secreted from Ob1771 cells is not active enough to induce the differentiation. (ii) The IGF-I mRNA is translated when cells are cultured in the presence of both GH and IGF-I, but not when cells are cultured in the presence of GH alone. (iii) The IGF-I protein is not secreted from Ob1771 cells without the signal from the IGF-I receptor. To examine these possibilities, we analyzed the expression of each class of IGF-I mRNA, and the expression and secretion of IGF-I protein during the induction of differentiation. Furthermore, we examined the translation efficiency of class 1 and class 1del. mRNAs in vitro. In this study we show that IGF-I in the medium changed the splicing pattern of IGF-I mRNA and allowed the synthesis of IGF-I protein.
To obtain conditioned media and cell lysates for Western blotting, cells were incubated in the serum free medium, ASF104 (Ajinomoto), for 24 h. After the medium was removed, the cells were harvested in the lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM EDTA, 0.5 mM (p-amidinophenyl)methanesulfonyl fluoride hydrochloride, 2 mM benzamidine, 165 KIU/ml aprotinin, and 1% Nonidet P-40.
Figure 2: Secretion and production of IGF-I. Ob1771 cells were cultured to confluence in the standard medium (day 0) and cultured up to day 4 in the differentiation medium supplemented with 10 nM GH (GH) or with 10 nM GH and 10 nM IGF-I (GH + IGF-I). The medium was changed to the standard medium, and the cells were cultured for 2 days. On the day indicated on top of the panel, the medium was changed to the serum-free medium, and the cells were cultured for 24 h. The cells and the media were collected separately and the IGF-I proteins in them were analyzed by Western blotting using the mouse anti-human IGF-I monoclonal antibody as described under ``Experimental Procedures.'' Similar results were obtained in three independent experiments.
Post-confluent Ob1771 cells were cultured for 2 days in the GH differentiation medium or in the GH-IGF-I differentiation medium followed by the maintenance for 1 day in the differentiation medium with no supplement, and a medium was finally obtained which was termed the GH conditioned medium or the GH-IGF-I conditioned medium, respectively. To analyze the activity of the secreted IGF-I, we examined the differentiation of another mass of Ob1771 cells with these two kinds of conditioned media based on the activation of GPDH. When the cells were cultured in the GH-IGF-I conditioned medium supplemented with 10 nM GH, the increment of the GPDH activity was 82% of that of fully differentiated cells cultured in the GH-IGF-I differentiation medium, and when the cells were cultured in the GH conditioned medium with GH, the increment of the GPDH activity was 17% of that of fully differentiated cells (data not shown). These results showed that the IGF-I protein secreted from Ob1771 cells had an activity that induced the differentiation of another mass of Ob1771 cells.
Figure 3: Translation efficiency of class 1 and class 1del. mRNAs in vitro. Class 1 mRNAs starting at 303 (lane 2) and 252 (lane 3) nucleotides upstream from the 3` end of exon1 and class 1del. mRNA (lane 4) were transcribed in vitro and in vitro translation of four micrograms each of mRNAs was performed in rabbit reticulocyte lysates containing biotin-Lys-tRNA. Biotinylated proteins were analyzed by Western blotting using horseradish peroxidase-labeled streptoavidin as described under ``Experimental Procedures.'' Lane 1 indicates a blank reaction without added mRNA. Similar results were obtained in three independent experiments.
In our studies, class 1 mRNA was proved to be more efficiently translated into protein than class 1del. mRNA in vitro. This suggests that exogenous IGF-I activates the intracellular production of IGF-I protein by shifting the splicing pattern of IGF-I mRNA from class 1del. to class 1, which is much more active in translation. The present result also suggests the presence of specific cis-elements involved in the translational control. It is reported that the efficiency of translation initiation is affected by the sequence context near the 5` cap(35) , by the upstream AUG codons and by the length and secondary structure of the mRNA leader(36) . Further studies are necessary to identify specific elements involved in the translational regulation of IGF-I mRNA. However, we cannot rule out the possibility that exogenous IGF-I also activates cellular translation machinery in vivo.
In Ob1771 cells, class 2 mRNA may be translationally inactive, since the time of its appearance and requirement of GH and IGF-I do not coincide with those for the IGF-I production. However, it is possible that class 2 mRNA is translationally regulated in different ways specified by cell types and growth stages.
We found that the GH-IGF-I conditioned medium, which contained IGF-I secreted from Ob1771 cells, had an activity that induced the differentiation of other Ob1771 cells. It is strongly suggested that the activity was attributed to IGF-I in the medium. However, we cannot rule out the possibility that another adipogenic factor is secreted from Ob1771 cells in the presence of GH combined with IGF-I and regulates the differentiation. Thus, we propose the following hypothesis. GH stimulates the transcription of the IGF-I gene, but the produced mRNA is mainly class 1del. mRNA, which is not efficiently translated into protein. Exogenous IGF-I acts on preadipocytes in an endocrine fashion to initiate the differentiation and also to initiate the synthesis and secretion of endogenous IGF-I, which then acts in an autocrine fashion and stimulates the next round of the production and secretion of endogenous IGF-I. Therefore once endogenous IGF-I was produced and secreted, exogenous IGF-I may not be needed any more in the medium. An Ob1771 cell stimulated by IGF-I combined with GH synthesizes and secretes many IGF-I molecules, which act on other Ob1771 cells in a paracrine fashion. Amplified IGF-I acts on a large number of cells and induces the differentiation and synthesis of IGF-I to a progressively greater extent. In this way, many cells can differentiate into adipocytes in response to the initial stimulation with IGF-I. Although it is not clear whether a similar mechanism regulates the expression of the IGF-I gene in other cell types, it is possible that one of the post-transcriptional control mechanisms of the IGF-I gene expression is the exogenous IGF-I-dependent regulation of alternative splicing of pre-mRNA