Dataset Information

Specific co-ordinated regulation of PC3 and PC2 gene expression with that of preproinsulin in insulin-producing beta TC3 cells.

ABSTRACT: Short-term (less than 2 h) glucose stimulation of isolated pancreatic islets specifically increases the biosynthesis of proinsulin and its converting enzymes PC2 and PC3 at the translation level. To determine whether gene expression of PC2 and PC3 was also regulated by longer-term (more than 6 h) glucose stimulation along with that of preproinsulin, studies were performed with the beta TC3 insulin-producing cell line. By Northern blot analysis, glucose maintained PC2 and PC3 mRNA levels in parallel with those of preproinsulin. After 48 h, mRNA levels of preproinsulin, PC2 and PC3 were, respectively, 2.9 (P < 0.05), 3.0 (P < 0.005) and 5.3 (P < 0.001) times greater in the presence of glucose than in beta TC3 cells cultured in the absence of glucose. Glucose-regulated PC2 and PC3 gene expression, like that of preproinsulin, was maximal at glucose concentrations above 5.5 mM. Studies of mRNA stability showed that the half-lives of PC2 (9 h) and PC3 (5 h) mRNA were much shorter than that of preproinsulin mRNA (over 24 h), but little effect of glucose on stability of these mRNAs was observed. Nuclear run-off analysis indicated that transcription of preproinsulin, PC2 and PC3 was modestly induced after 1 h exposure to 16.7 mM glucose. Therefore preproinsulin, PC2 and PC3 mRNA levels in beta TC3 cells were most probably maintained at the level of gene transcription. In contrast, elevation of cyclic AMP by forskolin had no effect on mRNA levels or gene transcription of preproinsulin, PC2 and PC3, despite a cyclic-AMP-induced phosphorylation of the cyclic AMP response element binding protein that correlated with a marked increase in cJun and cFos gene transcription in the same beta-cells. These results suggest that preproinsulin, PC2 and PC3 gene transcription can be specifically glucose-regulated in a mechanism that is unlikely to involve a key role for cyclic AMP. The co-ordinate increase in PC2 and PC3 mRNA levels with that of preproinsulin mRNA in response to chronic glucose represents a long-term means of catering for an increased demand on proinsulin conversion.

SUBMITTER: Schuppin GT

PROVIDER: S-EPMC1216892 | biostudies-other | 1996 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:PC2 and PC3 are subtilisin-like proteases involved in the maturation of prohormones and proneuropeptides within neuroendocrine cells. They are synthesized as zymogens that undergo autocatalytic maturation within the secretory pathway. Maturation of pro-PC2 is slow (t12 >8 h), exhibits a pH optimum of 5.5 and is dependent on calcium (K0.5 2 mM), while pro-PC3 maturation is relatively rapid (t12 15 min), exhibits a neutral pH optimum and is not calcium dependent. These differences in the rates and optimal conditions for activation of the proteases may contribute to the diversity of products generated by these proteases in different cell types. Although highly similar, there are two major differences between pro-PC2 and pro-PC3: the presence of an aspartate at position 310 in pro-PC2 compared with asparagine at the equivalent position in pro-PC3 (and all other members of the subtilisin family), and the N-terminal propeptides, which exhibit low sequence identity (30%). With a view to establishing the structural features that might be responsible for these differences in the maturation of pro-PC2 and pro-PC3, Asp310 in pro-PC2 was mutated to Asn, and Asn309 in pro-PC3 was mutated to Asp. Chimaeric proteins were also made consisting of the pro-region of PC2 fused to the mature portion of PC3 and the pro-region of PC3 fused to the mature region of PC2. The wild-type and mutant DNA constructs were then transcribed and translated in an in vitro system capable of supporting maturation of pro-PC2 and pro-PC3. The results demonstrated that Asp310 of pro-PC2 is responsible for the acidic pH optimum for maturation. Thus changing Asp310 to Asn shifted the pH optimum for maturation to pH 7.0. However, changing Asn309 of pro-PC3 to Asp had no effect on the optimum pH for maturation of pro-PC3. A chimaeric construct containing the propeptide of pro-PC2 attached to PC3 shifted the pH optimum for maturation from pH 7.0 to 6.0 and slowed down the rate of maturation (t12 >8 h). When attached to PC2, the pro-region of pro-PC3 had no effect on the optimum pH for maturation (pH 5.5-6.0), but it did accelerate the rate of maturation (t12 2 h). These results demonstrate that Asp310 and the pro-region of pro-PC2 contribute to the acidic pH optimum and low rate of maturation of this zymogen relative to its closely related homologue PC3.

Dataset Information

Specific co-ordinated regulation of PC3 and PC2 gene expression with that of preproinsulin in insulin-producing beta TC3 cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets