Cellular specificity of proexendin-4 processing in mammalian cells in vitro and in vivo.

Glucagon-like peptide-1 (GLP-1) is a potent stimulator of glucose-dependent insulin secretion. Exendin-4(1-39) (Ex-4), isolated from Gila monster venom, is a highly specific GLP-1 receptor agonist that exhibits a prolonged duration of action in vivo. Although the processing mechanisms underlying liberation of GLP-1 from its prohormone have been elucidated, those for Ex-4 remain unknown. To examine the requirements for proEx-4 processing in mammalian cells, BHK fibroblasts, InR1-G9 islet A cells, and AtT-20 corticotropes, which express different prohormone convertases (furin, prohormone convertase 2, and prohormone convertase 1, respectively) were transfected with full-length lizard proEx-4, and the processing of proexendin was examined by HPLC and RIA (n = 3). All of the transfected cell lines exhibited Ex-4-like immunoreactivity in the media, and Ex-4-like immunoreactivity was detected in extracts of InR1-G9 and AtT-20 cells. However, only media and extracts from AtT-20 cells (not InR1-G9 and BHK cells) contained a single peak by HPLC corresponding to synthetic Ex-4. To establish whether proEx-4 can be processed to Ex-4 in nonimmortalized mammalian cells in vivo, the molecular forms of exendin-4 were examined in mice expressing a metallothionein-proEx-4 transgene (n = 3-6 for both males and females). ProEx4 mRNA transcripts were detected by RT-PCR in a broad range of both endocrine and nonendocrine tissues. Ex-4-like immunoreactivity was detected in pituitary, fat, adrenals, and testes; however HPLC analyses demonstrated that processed Ex-4 was found only in adrenals and testes. These results indicate that lizard proEx-4 is processed to mature bioactive Ex-4 in both rodent endocrine and nonendocrine mammalian cell types in vitro and in murine tissues in vivo. These findings may be useful for engineering cells that express a lizard pro-Ex4 transgene for the treatment of type 2 diabetes.

Nerve-derived trophic factors and DNA elements controlling expression of genes encoding synaptic proteins in skeletal muscle fibers.

The neuromuscular junction represents an excellent model system for studying various critical issues in neurobiology at the molecular, cellular, and physiological levels. Our understanding of the basic events underlying synpase formation, maintenance, and plasticity has progressed considerably over the last few years primarily because of the numerous studies that have focused on this synapse and used sophisticated recombinant DNA technology. Recent data indicate that myonuclei located in the vicinity of the postsynaptic membrane are in a differential state of transcription compared to nuclei of the extrasynaptic sarcoplasm. Thus, renewal of postsynaptic membrane proteins appears to occur via a mechanism involving the local transcriptional activation of genes encoding these specialized proteins and extracellular cues originating from motoneurons. Such interaction between presynaptic nerve terminals and the postsynaptic sarcoplasm indicates that the entire signal transduction pathway is compartmentalized at the level of the neuromuscular junction. Expression of these genes appears less coregulated than originally anticipated, indicating that maintenance of the postsynaptic membrane requires the contribution of multiple extracellular signals, which ultimately urge target transcription factors to distinct DNA regulatory elements via various second messenger systems.

Increased expression of acetylcholinesterase T and R transcripts during hematopoietic differentiation is accompanied by parallel elevations in the levels of their respective molecular forms.

Differentiation of hematopoietic cells is known to be accompanied by profound changes in acetylcholinesterase (AChE) enzyme activity, yet the basic mechanisms underlying this developmental regulation remain unknown. We initiated a series of experiments to examine the molecular mechanisms involved in regulating AChE expression during hematopoiesis. Differentiation of murine erythroleukemia (MEL) cells using dimethyl sulfoxide resulted in a 5- and 10-fold increase in intracellular and secreted AChE enzyme activity, respectively. Interestingly, these increases resulted from a preferential induction of the globular molecular form G1 and a slight increase in G4 instead of an increase in the levels of the G2 membrane-bound form, a molecular form expressed in mature erythrocytes. Concomitantly, expression of the two predominant AChE transcripts (R and T, for read-through and tail, respectively) in MEL cells was induced to a similar extent with differentiation. Nuclear run-on assays performed with nuclei isolated from induced versus uninduced MEL cells revealed that in contrast to the large increases seen in the transcription of the beta-globin gene, the transcriptional activity of the AChE gene remained largely unaffected after differentiation. Determination of the half-lives of the R and T transcripts demonstrated that they both exhibited an increase in stability in induced MEL cells. Taken together, results from these studies indicate that post-transcriptional regulatory mechanisms account for the increased expression of AChE in differentiated hematopoietic cells.