Abstract
Using a zebrafish model of hepatoerythropoietic porphyria (HEP), we identify a previously unknown mechanism underlying heme-mediated regulation of exocrine zymogens. Zebrafish bach1b, nrf2a and mafK are all expressed in the zebrafish exocrine pancreas. Overexpression of bach1b or knockdown of nrf2a result in the downregulation of the expression of the exocrine zymogens, whereas overexpression of nrf2a or knockdown of bach1b cause their upregulation. In vitro luciferase assays demonstrate that heme activates the zymogens in a dosage-dependent manner and that the zymogen promoter activities require the integral Maf recognition element (MARE) motif. The Bach1b-MafK heterodimer represses the zymogen promoters, whereas the Nrf2a-MafK heterodimer activates them. Furthermore, chromatin immunoprecipitation (ChIP) assays show that MafK binds to the MARE sites in the 5′ regulatory regions of the zymogens. Taken together, these data indicate that heme stimulates the exchange of Bach1b for Nrf2a at MafK-occupied MARE sites and that, particularly in heme-deficient porphyria, the repressive Bach1b-MafK heterodimer dominates, which can be exchanged for the activating Nrf2a-MafK heterodimer upon treatment with hemin. These results provide novel insights into the regulation of exocrine function, as well as the pathogenesis of porphyria, and should be useful for designing new therapies for both types of disease.
Highlights
As the prosthetic moiety for numerous proteins and enzymes – such as hemoglobin, catalases and cytochrome – heme is essential for most forms of life through oxygen transport, respiration, detoxification and other important processes (Padmanaban et al, 1989; Igarashi and Sun, 2006; Mense and Zhang, 2006; Balwani and Desnick, 2012)
They show that heme determines pancreatic zymogen production levels by mediating the dynamic exchange of the basic leucine zipper (bZIP) transcription factors Bach1b and Nrf2a at musculoaponeurotic fibrosarcoma (Maf) recognition element (MARE) sites that are occupied by MafK, another bZIP transcription factor
The authors show that treatment with hemin can dissociate Bach1b from MafK, which allows Nrf2a to enter the MafK-occupied MARE sites
Summary
As the prosthetic moiety for numerous proteins and enzymes – such as hemoglobin, catalases and cytochrome – heme is essential for most forms of life through oxygen transport, respiration, detoxification and other important processes (Padmanaban et al, 1989; Igarashi and Sun, 2006; Mense and Zhang, 2006; Balwani and Desnick, 2012). Our microarray and in situ hybridization analyses of this zebrafish HEP model revealed downregulation of six peptidase precursor genes – including carboxypeptidase A5 (cpa5), chymotrypsinogen 1 like (ctr1l), chymotrypsinogen B1 (ctrb1), elastase 2 like (ela2l), trypsin precursor (try) and trypsin like (tryl) – in the exocrine pancreas of the HEP zebrafish (yquem/urod, −/−) (Wang et al, 2007) Of these six zymogens, cpa (previously called cpa) belongs to the MEROPS peptidase family M14, whereas the other five peptidases contain a trypsin-like serine protease (tryp_SPc) domain and are members of the serine peptidase chymotrypsin family S1 (chymotrypsin A, clan PA) (http://merops.sanger.ac.uk/) (Wang et al, 2007). The molecular mechanism underlying how these exocrine zymogens regulate heme remains poorly understood (Wang et al, 2007)
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