Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons.

N-Bak is a neuron-specific BH3-only splice variant of pro-apoptotic Bcl-2 family member Bak. We have shown that its mRNA is stable in the neurons, whereas the protein cannot be detected by antibodies, suggesting a strong translational arrest of the mRNA. Here we identify two regulatory elements in the N-Bak mRNA that significantly repress translation in the luciferase reporter assay: an upstream open reading frame in the 5'-untranslated region (UTR) and naturally spliced exon-exon junction downstream of the premature translation termination codon in the 3'UTR. We also show that N-Bak mRNA is stored in granular structures in the sympathetic neurons and stays in these granules during intrinsic apoptosis. Finally, we confirm the absence of N-Bak protein by quantitative mass spectrometry analysis in the healthy, apoptotic or stressed sympathetic and cortical neurons. We conclude that N-Bak mRNA is translationally repressed by multiple mechanisms, and the protein does not participate in the classical apoptosis or cellular stress response.

Epidermal growth factor (EGF)-receptor signalling is needed for murine beta cell mass expansion in response to high-fat diet and pregnancy but not after pancreatic duct ligation.

AIMS/HYPOTHESIS: Epidermal growth factor receptor (EGFR) signalling is essential for the proper fetal development of pancreatic islets and in the postnatal formation of an adequate beta cell mass. In this study we investigated the role of EGFR signalling in the physiological states of beta cell mass expansion in adults during metabolic syndrome and pregnancy, as well as in regeneration after pancreatic duct ligation. METHODS: Heterozygous Pdx1-EGFR-dominant-negative (E1-DN) mice, which have a kinase-negative EGFR under the Pdx1 promoter, and wild-type mice were both subjected to a high-fat diet, pregnancy and pancreatic duct ligation. RESULTS: The beta cell mass of wild-type mice fed the high-fat diet increased by 70% and the mice remained normoglycaemic; the E1-DN mice became diabetic and failed to show any compensatory beta cell mass expansion. Similarly, pregnant wild-type mice had four times more proliferating beta cells and a 75% increase in beta cell mass at mid-gestation, in contrast to the pregnant E1-DN mice, which did not show any significant beta cell compensation and were hyperglycaemic in an intraperitoneal glucose tolerance test. However, after pancreatic duct ligation, both the wild-type and E1-DN mice showed similar expression of Ngn3 (also known as Neurog3) and beta cell proliferation increased to a similar level in the ligated part of pancreas. CONCLUSIONS/INTERPRETATIONS: EGFR signalling is essential in beta cell mass expansion during a high-fat diet and pregnancy where replication is the primary mechanism for compensatory beta cell mass expansion. In contrast, EGFR signalling appears not to be crucial to increased beta cell proliferation after pancreatic duct ligation.

Blood vessels of human islets of Langerhans are surrounded by a double basement membrane.

AIMS/HYPOTHESIS: Based on mouse study findings, pancreatic islet cells are supposed to lack basement membrane (BM) and interact directly with vascular endothelial BM. Until now, the BM composition of human islets has remained elusive. METHODS: Immunohistochemistry with specific monoclonal and polyclonal antibodies as well as electron microscopy were used to study BM organisation and composition in human adult islets. Isolated islet cells and function-blocking monoclonal antibodies and recombinant soluble Lutheran peptide were further used to study islet cell adhesion to laminin (Lm)-511. Short-term cultures of islets were used to study Lutheran and integrin distribution. RESULTS: Immunohistochemistry revealed a unique organisation for human Lm-511/521 as a peri-islet BM, which co-invaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained Lm-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the Lm alpha5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR, whereas alpha(3) and beta(1) integrins were more diffusely distributed. High Lutheran content was also found on endocrine cell membranes in short-term culture of human islets. The adhesion of dispersed beta cells to Lm-511 was inhibited equally effectively by antibodies to integrin and alpha(3) and beta(1) subunits, and by soluble Lutheran peptide. CONCLUSIONS/INTERPRETATION: The present results disclose a hitherto unrecognised BM organisation and adhesion mechanisms in human pancreatic islets as distinct from mouse islets.

Nephrin is expressed in the pancreatic beta cells.

AIMS/HYPOTHESIS: The NPHS1 gene product, nephrin, is a crucial component of the glomerular filtration barrier preventing proteinuria and previously assumed to be kidney-specific. The aim of this study was to describe the expression of nephrin mRNA and protein in human pancreas as well as identify the nephrin-expressing cell types. METHODS: RNA dot blot, reverse transcriptase-polymerase chain reaction, sequencing, immunoblotting and dual immunofluorescence were used for the characterisation of nephrin in the pancreas. RESULTS: Except for the kidney, the pancreas was found to be the only tissue expressing nephrin as screened with a human tissue RNA dot blot. The expression was verified with reverse transcriptase-polymerase chain reaction and by sequencing nephrin from a human pancreatic complementary DNA library. Nephrin antibody in immunoblot detected a 165,000 M(r) protein in the pancreas. Dual immunofluorescence showed that nephrin was specifically localised in the beta cells of the islets of Langerhans. There was no overlap with glucagon, somatostatin, or the ductal cell marker cytokeratin 19. CONCLUSION/INTERPRETATION: These data show that nephrin is a novel molecule of pancreatic beta cells.

Transcription factor expression and hormone production in pancreatic AR42J cells.

AR42J is an exocrine pancreatic cell line that has been reported to differentiate towards an endocrine phenotype when stimulated with various growth factors, such as activin A, hepatocyte growth factor (HGF), betacellulin or glucagon-like peptide 1. In our experiments, AR42J-B13 cells differentiated morphologically in response to the growth factor treatment as reported previously. However, they failed to express the insulin gene. We found that the cells did not express several transcription factors known to be found in the beta-cell, including Nkx6.1, isl-1, Pax4 and Pax6. In addition, the mRNA level for pdx-1 and Nkx2.2 were very low in comparison to the insulinoma cell lines INS-1 and RINm5F. However, some transcription factors typically found in beta-cells and neuroendocrine cells were expressed also in the AR42J-B13 cells. These included BETA2/NeuroD, HNF1alpha, C/EBPbeta and IA-1. Unlike the insulinoma cells, AR42J cells expressed the exocrine transcription factor p48. In order to induce endocrine differentiation, we transfected the AR42J-B13 cells with the full length cDNAs of isl-1, Nkx6.1, Nkx2.2 and pdx-1 under the control of the CMV promoter, both separately and in combinations. The expression of Nkx2.2 led consistently to the appearance of pancreatic polypeptide but not insulin, glucagon or somatostatin mRNA. The PP mRNA expression in Nkx2.2 cDNA transfected cells was independent of the growth factor treatment used for differentiating AR42J cells. In conclusion, the AR42J-B13 line possesses some features of a pancreatic neuroendocrine cell. However, we were unable to confirm the capacity of these cells to differentiate into insulin-producing cells. Our results indicate that Nkx2.2 plays a role in the transcriptional regulation of PP expression.

Impaired migration and delayed differentiation of pancreatic islet cells in mice lacking EGF-receptors.

Pancreatic acini and islets are believed to differentiate from common ductal precursors through a process requiring various growth factors. Epidermal growth factor receptor (EGF-R) is expressed throughout the developing pancreas. We have analyzed here the pancreatic phenotype of EGF-R deficient (-/-) mice, which generally die from epithelial immaturity within the first postnatal week. The pancreata appeared macroscopically normal. The most striking feature of the EGF-R (-/-) islets was that instead of forming circular clusters, the islet cells were mainly located in streak-like structures directly associated with pancreatic ducts. Based on BrdU-labelling, proliferation of the neonatal EGF-R (-/-) beta-cells was significantly reduced (2.6+/-0.4 versus 5.8+/-0.9%, P<0.01) and the difference persisted even at 7-11 days of age. Analysis of embryonic pancreata revealed impaired branching morphogenesis and delayed islet cell differentiation in the EGF-R (-/-) mice. Islet development was analyzed further in organ cultures of E12.5 pancreata. The proportion of insulin-positive cells was significantly lower in the EGF-R (-/-) explants (27+/-6 versus 48+/-8%, P<0.01), indicating delayed differentiation of the beta cells. Branching of the epithelium into ducts was also impaired. Matrix metalloproteinase (MMP-2 and MMP-9) activity was reduced 20% in EGF-R (-/-) late-gestation pancreata, as measured by gelatinase assays. Furthermore, the levels of secreted plasminogen activator inhibitor-1 (PAI-1) were markedly higher, while no apparent differences were seen in the levels of active uPA and tPa between EGF-R (-/-) and wild-type pancreata. Our findings suggest that the perturbation of EGF-R-mediated signalling can lead to a generalized proliferation defect of the pancreatic epithelia associated with a delay in beta cell development and disturbed migration of the developing islet cells as they differentiate from their precursors. Upregulated PAI-1 production and decreased gelatinolytic activity correlated to this migration defect. An intact EGF-R pathway appears to be a prerequisite for normal pancreatic development.

Growth factor-mediated proliferation and differentiation of insulin-producing INS-1 and RINm5F cells: identification of betacellulin as a novel beta-cell mitogen.

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.

Biosynthesis of heparin/heparan sulfate. cDNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung.

Glucuronyl C5-epimerases catalyze the conversion of D-glucuronic acid (GlcUA) to L-iduronic acid (IdceA) units during the biosynthesis of glycosaminoglycans. An epimerase implicated in the generation of heparin/heparan sulfate was previously purified to homogeneity from bovine liver (Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén, L., Lindahl, U., and Li, J.-p. (1994) J. Biol. Chem. 269, 26953-26958). The present report describes the molecular cloning and functional expression of the lung enzyme. The cloned enzyme contains 444 amino acid residues and has a molecular mass of 49,905 Da. N-terminal sequence analysis of the isolated liver enzyme showed this species to be a truncated form lacking a 73-residue N-terminal domain of the deduced amino acid sequence. The coding cDNA insert was cloned into a baculovirus expression vector and expressed in Sf9 insect cells. Cells infected with recombinant epimerase showed a 20-30-fold increase in enzyme activity, measured as release of 3H2O from a polysaccharide substrate containing C5-3H-labeled hexuronic acid units. Furthermore, incubation of the expressed protein with the appropriate (GlcUA-GlcNSO3)n substrate resulted in conversion of approximately 20% of the GlcUA units into IdceA residues. Northern analysis implicated two epimerase transcripts in both bovine lung and liver tissues, a dominant approximately 9-kilobase (kb) mRNA and a minor approximately 5-kb species. Mouse mastocytoma cells showed only the approximately 5-kb transcript. A comparison of the cloned epimerase with the enzymes catalyzing an analogous reaction in alginate biosynthesis revealed no apparent amino acid sequence similarity.

Induced expression of neurotrophins in transgenic mice overexpressing ornithine decarboxylase and overproducing putrescine.

Transgenic mice overexpressing ornithine decarboxylase (ODC) were recently generated (Halmekytö et al.: Biochem Biophys Res Commun 180:262-267, 1991). The dramatic ODC overexpression resulted in a very high accumulation of the polyamine putrescine in the brain. As elevated polyamine levels in the brain are believed to be associated with neuronal damage, we studied whether enhanced putrescine accumulation in the brain of these mice affects the expression of neurotrophins and their high affinity receptors. Northern blot analysis indicated that mRNA levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) were significantly elevated about 1.5-fold in the hippocampus of ODC transgenic mice as compared with control animals. The levels of BDNF, NGF and NT-3 mRNA were also elevated in the kidneys of the transgenic mice. In eight other tissues examined there were no significant differences. The expression pattern of BDNF mRNA and of trkB and trkC (high affinity receptors for BDNF and NT-3 respectively) immunoreactivity in the hippocampal formation did not reveal significant differences. The induction of the expression of neurotrophins could belong to neuroprotective measures triggered by ODC overexpression.

Neurotrophin 3 rescues neuronal precursors from apoptosis and promotes neuronal differentiation in the embryonic metanephric kidney.

We analyzed the developmental regulation and role of the neurotrophins during metanephric kidney morphogenesis. RNase protection assay revealed the presence of nerve growth factor, neurotrophin 3 (NT-3), and brain-derived neurotrophic factor mRNAs and the regulation of their expression during embryonic development of rat metanephros. NT-3 induced differentiation (neurite outgrowth) and survival (inhibition of apoptosis) of the neuronal precursors in cultured nephrogenic mesenchymes and neuronal differentiation in cultured whole kidneys, whereas NT-4/5, brain-derived neurotrophic factor, and nerve growth factor were without effect. The neurotrophins did not trigger tubular differentiation of isolated nephrogenic cells, which underwent apoptosis when cultured with or without the neurotrophins. NT-3 is thus an inducer of differentiation and a survival factor for renal neuronal cells, but none of the neurotrophins is a morphogen in kidney tubule induction.

Characterization of the rat light neurofilament (NF-L) gene promoter and identification of NGF and cAMP responsive regions.

We have isolated a genomic DNA clone covering the coding and 14 kb upstream region of the rat light neurofilament (NF-L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF-L promoter, constructs carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT) reporter gene were tested for their capability to direct CAT expression after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between -407 and +75 bp from the transcription initiation site. The NGF-responsive element was located between -38 and +75 bp, and sequence -97 to -38 was found to contain a functional cAMP-responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT expression was stimulated up to 12-fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF-L promoter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed.

Coordinated expression and function of neurotrophins and their receptors in the rat inner ear during target innervation.

We show that trkB and trkC mRNAs, encoding the high-affinity receptor tyrosine kinases for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), respectively, as well as low-affinity nerve growth factor receptor (p75LNGFR) mRNA are expressed in the cochleovestibular ganglion (CVG) before and during innervation of the target fields. Correspondingly, from preinnervation stages onward, BDNF and NT-3, but neither nerve growth factor (NGF) nor neurotrophin-4 (NT-4) mRNAs are expressed in the sensory epithelium of the otic vesicle, the peripheral target field of CVG neurons. No neurotrophin transcripts were detected by in situ hybridization in the medullary central targets. In explant cultures, neuritogenesis from both the cochlear and vestibular part of the CVG was promoted by BDNF, while NT-3 evoked neurites mainly from the cochlear neurons. Also NT-4 stimulated neurite outgrowth from the CVG in vitro. In dissociated neuron-enriched cultures, NT-3 and BDNF promoted survival of overlapping subsets of CVG neurons and, correspondingly, results from in situ hybridization showed that both trkC and trkB mRNAs were expressed in most neurons of this ganglion. The negligible effect of NGF seen in the bioassays agrees well with the expression of only a few trkA transcripts, encoding the high-affinity receptor for NGF, in the CVG. Based on the spatiotemporal expression patterns and biological effects in vitro, peripherally-synthesized BDNF and NT-3 regulate the survival of CVG neurons as well as the establishment of neuron-target cell contacts in the early-developing inner ear. In addition, the expression of trkB mRNA, more specifically its truncated form, and trkC as well as p75LNGFR mRNAs in distinct non-neuronal structures indicates novel roles for these molecules during development.

Neuronal characteristics in embryonic renal stroma.

The metanephric mesenchyme is considered a homogeneous population of predetermined, but pluripotent cells with a nephrogenic bias. By an inductive stimulus, the mesenchyme is programmed to differentiate into the various epithelial phenotypes of the secretory nephron. A fraction of the mesenchymal cells, however, remains in the interstitium between the nephrons and differentiates into spindle-shaped, clear-cytoplasmic renal stroma. We have analyzed the molecular nature of these cells in order to discover the specific cell types that could be involved in the morphogenetic processes during kidney differentiation. In situ hybridization reveals neurofilament light protein mRNA, and immunohistology shows neurofilament light and medium proteins in the stromal cells around kidney tubules. By immunohistochemistry these peritubular stromal cells can be distinguished from the neuronal cells of the renal microganglion: the peritubular stromal cells are neurofilament-positive but L1 neural cell adhesion protein-negative, whereas the neuronal cells with axonal extension are both neurofilament-positive and L1 neural cell adhesion protein-positive. Proliferation index of the stromal cells was low as compared to tubular cells, as shown by bromodeoxyuridine incorporation.

Neurotrophins and their receptors in rat peripheral trigeminal system during maxillary nerve growth.

We examined the expression of the neurotrophins (NTFs) and their receptor mRNAs in the rat trigeminal ganglion and the first branchial arch before and at the time of maxillary nerve growth. The maxillary nerve appears first at embryonic day (E)10 and reaches the epithelium of the first branchial arch at E12, as revealed by anti-L1 immunohistochemistry. In situ hybridization demonstrates, that at E10-E11, neurotrophin-3 (NT-3) mRNA is expressed mainly in the mesenchyme, but neurotrophin-4 (NT-4) mRNA in the epithelium of the first branchial arch. NGF and brain-derived neurotrophic factor (BDNF) mRNAs start to be expressed in the distal part of the first brachial arch shortly before its innervation by the maxillary nerve. Trigeminal ganglia strongly express the mRNA of trkA at E10 and thereafter. The expression of mRNAs for low-affinity neurotrophin receptor (LANR), trkB, and trkC in trigeminal ganglia is weak at E10, but increases by E11-E12. NT-3, NT-4, and more prominently BDNF, induce neurite outgrowth from explant cultures of the E10 trigeminal ganglia but no neurites are induced by NGF, despite the expression of trkA. By E12, the neuritogenic potency of NGF also appears. The expression of NT-3 and NT-4 and their receptors in the trigeminal system prior to target field innervation suggests that these NTFs have also other functions than being the target-derived trophic factors.

Expression patterns of neurotrophin and their receptor mRNAs in the rat inner ear.

In situ hybridization was used to study the expression of mRNAs of nerve growth factor (NGF), brain-derived neutrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-5 (NT-5) and the components of their high-affinity receptors in the early postnatal and adult rat inner ears. NGF or NT-5 transcripts were not detected in the inner ear neuroepithelium or in the innervating neurons. NT-3 mRNA was intensely expressed over the one-week-old and adult inner hair cells (IHCs) but in the outer hair cells (OHCs) and vestibular maculae only during the early postnatal period. BDNF mRNA was expressed in the IHCs and OHCs of the early postnatal cochlea but not in the adult organ of Corti. High levels of BDNF transcripts were observed in the sensory epithelia of all vestibular end organs. mRNAs of low affinity NGF receptor, trkB and trkC, but not of trk, were expressed in the spiral and vestibular ganglia. In addition, the non-catalytic form of trkB mRNA localized to the sensory epithelia of maculae utriculi and sacculi. The present results show that of the neurotrophins examined, NT-3 is the predominant neurotrophin in the adult organ of Corti and BDNF is that in vestibular organs. The expression patterns of NT-3 and BDNF mRNAs suggest that these neurotrophins may participate in the maintenance of mature cochleovestibular neurons and they may be involved in the survival response of injured neurons.

Brain-derived neurotrophic factor and neurotrophin 3 mRNAs in the peripheral target fields of developing inner ear ganglia.

In situ hybridization was used to study the site and timing of the expression of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 5 (NT-5) mRNAs in the developing inner ear of the rat. In the sensory epithelia, the levels of NGF and NT-5 mRNAs were below the detection limit. NT-3 and BDNF mRNAs were expressed in the otic vesicle in overlapping but also in distinct regions. Later in development, NT-3 transcripts were localized to the differentiating sensory and supporting cells of the auditory organ and vestibular maculae. In these sensory epithelia, the intensity of NT-3 mRNA expression decreased in parallel with maturation. The expression of BDNF mRNA was restricted to the sensory cells of both the auditory and vestibular organs, including ampullary cristae. In bioassays, BDNF and NT-3, but not NGF, at physiological concentrations induced neurite outgrowth from the statoacoustic ganglion explants. These results demonstrate that NT-3 and BDNF, rather than NGF and NT-5, are the primary neurotrophins present in the target fields of the cochlear and vestibular neurons. Expression of NT-3 and BDNF mRNAs in the otic vesicle before and during the ingrowth of neurites from the statoacoustic ganglion suggests that NT-3 or BDNF or both may serve as chemoattractants for the early nerve fibers. The results also suggest that these neurotrophins have a role in later development of the cochlear and vestibular neurons.

Dependence of kidney morphogenesis on the expression of nerve growth factor receptor.

Nerve growth factor receptor (NGFR) serves as the binding site for the neurotrophic growth factors. Although NGFR has been found in several embryonic tissues outside the nervous system, the function of NGFR in embryogenesis of non-neuronal organs remains unknown. NGFR is transiently synthesized by embryonic rat kidney and disappears from nephrons upon their terminal differentiation. Anti-sense oligonucleotide inhibition of NGFR expression inhibits kidney morphogenesis. Therefore, NGFR is required not only for development of the nervous system, but also for differentiation of the kidney tubules.