The neuregulin (NRG)/epidermal growth factor (EGF) family of growth factors consists of several ligands that specifically activate four erbB receptor-tyrosine kinases, namely erbB-1 (EGF-R), erbB-2 (neu), erbB-3, and erbB-4. We have previously shown that islet morphogenesis is impaired and beta-cell differentiation delayed in mice lacking functional EGF-R [EGF-R (-/-)]. The present study aims to clarify which erbB ligands are important for islet development. Pancreatic expression of EGF, TGF-alpha, heparin-binding EGF, betacellulin (BTC), and NRG-4 was detected as early as embryonic d 13 (E13). Effects of these ligands were studied in E12.5 pancreatic explant cultures grown for 5 d ex vivo. None of the growth factors affected the ratio of endocrine to exocrine cells. However, significant effects within the endocrine cell populations were induced by EGF, BTC, and NRG-4. beta-Cell development was augmented by BTC, whereas the development of somatostatin-expressing delta-cells was stimulated by NRG-4. Both ligands decreased the numbers of glucagon-containing alpha-cells. The effect of BTC was abolished in the EGF-R (-/-) mice. A soluble erbB-4 binding fusion protein totally inhibited the effects of NRG-4 but not of BTC. Neutralization of endogenous NRG-4 activity in the model system effectively inhibited delta-cell development, indicating that this erbB4-ligand is an essential factor for delineation of the somatostatin-producing delta-cells. Our results suggest that ligands of the EGF-R/erbB-1 and erbB-4 receptors regulate the lineage determination of islet cells during pancreatic development. BTC, acting through EGF-R/erbB-1, is important for the differentiation of beta-cells. This could be applied in the targeted differentiation of stem cells into insulin-producing cells.

Transforming growth factor-β (TGF-β) superfamily related growth factors signal by binding to transmembrane type I and type II receptor serine/threonine kinases (RSTK), which phosphorylate intracellular Smad transcription factors in response to ligand binding. Here we describe the cloning of the human type I RSTK activin receptor-like kinase 7 (ALK7), an orthologue of the previously identified rat ALK7. Nodal, a TGF-β member expressed during embryonic development and implicated in developmental events like mesoderm formation and left-right axis specification, was recently shown to signal through ALK7. We found ALK7 mRNA to be most abundantly expressed in human brain, pancreas and colon. A cDNA encoding the open reading frame of ALK7 was obtained from a human brain cDNA library. Furthermore, a P1 artificial chromosome (PAC) clone containing the human ALK7 gene was isolated and fluorescent in situ hybridization (FISH) on metaphase chromosomes identified the gene locus as chromosome 2q24.1→q3. To test the functionality of the ALK7 signaling, we generated recombinant adenoviruses containing a constitutively active form of ALK7 (Ad-caALK7), which is capable of activating downstream targets in a ligand independent manner. Infection with Ad-caALK7 of MIN6 insulinoma cells, in which ALK7 has previously been shown to be endogenously expressed, led to a marked increase in the phosphorylation of Smad2, a signaling molecule also used by TGF-βs and activins.

I slet transplantation represents a potential treatment for insulin-dependent diabetes. Development and application of this technique is hampered by the limited availability of human donor tissue. In vitro expansion of human p-cells may provide an adequate source for p-cell grafts. So far, attempts to expand adult rodent p-cells have been unsuccessful. In different experimental conditions, this cell type has a low rate of replication both in vivo and in vitro (1,2). Nutrients and growth factors can stimulate fetal or neonatal P-cell replication, but most adult p-cells appear unresponsive to growth factor stimulation in vitro (1). In Address correspondence and reprint requests to Veronique H. Lefebvre, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail: velefeb@mebo.vub.ac.be.Received for publication 29 August 1997 and accepted in revised form 9 October 1997.BrdU, 5-bromo-2'-deoxyuridine; HGF, hepatocyte growth factor .vivo, the main mechanism leading to increased p-cell numbers appears to involve neogenesis from ductal progenitor cells (2,3). Human p-cells exhibit a very low proliferative activity both during fetal development (9) and in adult life (4). According to a recent report, the proliferation of fetal human p-cells can be stimulated by hepatocyte growth factor (HGF) (5). Furthermore, also adult human p-cells appear to proliferate when cultured in the presence of HGF and an extracellular matrix prepared from rat 804G cells (6). The present study compares the mitogenic effect of this condition on the different cell types that compose a human islet preparation. Our findings contradict the conclusion of Hayek et al. (6), but indicate, instead, that HGF stimulates the proliferation of adult pancreatic duct cells. RESEARCH DESIGN AND METHODSIslet isolation and culture. Human islets from ten heart-beating organ donors were isolated at the Central Unit of p-cell Transplant, Medical Campus, Vrije Universiteit, Brussels. The mean age of the organ donors was 41 ± 5 years (means ± SE [range 19-64]) and the mean organ preservation time 11 ± 2 h (range 4-20 h). Pancreases were processed by ductal distension with collagenase, gentle dissociation, and Ficoll gradient purification of islets. Islet-enriched fractions were cultured in serum-free Ham's F10 medium (Gibco, Life Technologies, Paisley, Scotland, U.K.) with 7.5 mmol/1 glucose, 1% bovine serum albumin, 0.075 ing penicillin/ml, 0.1 mg streptomycin/ml, and 2 mmol/1 glutamine (7). After 4-16 days of culture, these preparations contained <5% damaged cells, no acinar cells, 25 ± 5% ductal cells, 63 ± 5% insulin-positive cells, and 12 ± 2% glucagon-positive cells. They were recovered within this culture period and distributed into 35-mm dishes for either suspension culture or monolayer culture on 804G matrix (6) for a subsequent 1-7 days culture in RPMI1640 medium (Gibco, Life Technologies) with 5.5 mmol/1 or 11.1 mmol/1 glucose, 10% pooled AB human serum (BioWhittaker, Walkersville, MD, or Finnish Red Cross, Helsin...

It is not clear which growth factors are crucial for the survival, proliferation, and differentiation of pancreatic beta-cells. We used the relatively differentiated rat insulinoma cell line INS-1 to elucidate this issue. Responsiveness of the DNA synthesis of serum-starved cells was studied to a wide variety of growth factors. The most potent stimulators were PRL, GH, and betacellulin, a member of the epidermal growth factor (EGF) family that has not previously been shown to be mitogenic for beta-cells. In addition to these, only vascular endothelial growth factor, insulin-like growth factor-1 and -2, had significant mitogenic activity, whereas hepatocyte growth factor, nerve growth factor-beta, platelet-derived growth factors, basic fibroblast growth factor, EGF, transforming growth factor-alpha (TGF-alpha), neu differentiation factor, and TGF-beta were inactive. None of these factors affected the insulin content of INS-1 cells. In contrast, certain differentiation factors, including nicotinamide, sodium butyrate, activin A, and 1,25-dihydroxyvitamin D3 inhibited the DNA synthesis and increased the insulin content. Also all-trans-retinoic acid had an inhibitory effect on cell DNA synthesis but no effect on insulin content. From these findings betacellulin emerges as a novel growth factor for the beta-cell. Half-maximal stimulation of INS-1 DNA synthesis was obtained with 25 pM betacellulin. Interestingly, betacellulin had no effect on RINm5F cells, whereas both EGF and TGF-alpha were slightly mitogenic. These effects may possibly be explained by differential expression of the erbB receptor tyrosine kinases. In RINm5F cells a spectrum of erbB gene expression was detected (EGF receptor/erbB-1, erbB-2/neu, and erbB-3), whereas INS-1 cells showed only expression of EGF receptor. Expression of the erbB-4 gene was undetectable in these cell lines. In summary, our results suggest that the INS-1 cell line is a suitable model for the study of beta-cell growth and differentiation because the responses to previously identified beta-cell mitogens were essentially similar to those reported in primary cells. In addition, we have identified betacellulin as a possible modulator of beta-cell growth.