Hepatic Gene Expression in Protoporphyic Fech Mice Is Associated with Cholestatic Injury but Not a Marked Depletion of the Heme Regulatory Pool

Abstract

BALB/c Fech m1Pas mice have a mutated ferrochelatase gene resulting in protoporphyria that models the hepatic injury occurring sporadically in human erythropoietic protoporphyria. We used this mouse model to study the development of the injury and to compare the dysfunction of heme synthesis with hepatic gene expression of liver metabolism, oxidative stress, and cellular injury/inflammation. From an early age expression of total cytochrome P450 and many of its isoforms was significantly lower than in wild-type mice. However, despite massive accumulation of protoporphyrin in the liver, expression of the main genes controlling heme synthesis and catabolism Heme is vital to the transport and utilization of oxygen in oxidative and signaling pathways. A large proportion of heme usage in the liver is accounted for by cytochrome P450 isoforms, many of which are associated with drug metabolism. In the final step of heme synthesis ferrous iron is inserted into the precursor porphyrin protoporphyrin IX by the mitochondrial enzyme ferrochelatase. The structure and regulation of ferrochelatase have been extensively studied.

Neurite Degeneration Induced by Heme Deficiency Mediated via Inhibition of NMDA Receptor-Dependent Extracellular Signal-Regulated Kinase 1/2 Activation

“…The BALB/c Fech m1Pas mouse strain was obtained from the The Jackson Laboratory (Bar Harbor, ME). The Fech m1Pas mutant contains a point mutation in the ferrochelatase gene (Tutois et al, 1991;Davies et al, 2005). Mice were bred by homozygous mating and maintained in a negative pressure isolator at 21°C under reduced light to prevent skin lesions.…”

“…Proteins were extracted from primary neurons after 14 and 21 d of culturing using lysis buffer (7 M urea, 50 mM Tris-HCl, pH7.5, and 5 mM DTT) followed by brief sonication. SDS electrophoresis and immunoblotting were performed (Davies et al, 2005) using enhanced chemiluminescence detection (GE Healthcare, Little Chalfont, UK) and primary antibodies from the following sources: phospho-ERK1/2, ERK1/2, phospho-c-Raf (Ser338), phospho-c-Raf (Ser259), phospho-NMDAR1 (Ser 890, Ser 896, and Ser 897), phospho-JNK (T183/185), phospho-p38 mitogen-activated protein kinase (MAPK), phospho-cAMP response element-binding protein (CREB), and total CREB from Cell Signaling Technology (Beverly, MA); NMDA ⌵R1, ␣-tubulin, B-Raf, and phospho-B-Raf from Santa Cruz Biotechnology (Santa Cruz, CA); and NMDA NR2B and phospho-NMDA NR2B from PhosphoSolutions (Aurora, CO). Results were quantified using densitometry and Image Quant 5.2 software.…”

Multiple polymorphic loci determine basal hepatic and splenic iron status in mice

“…Tissues from mice for histological and immunohistochemical analyses were fixed in buffered formalin. 29 Antimouse DCYTB11-A peptide antibody was from Alpha Diagnostic (San Antonio, TX).…”

“…resgen.com) and the IMAGE collection and analyses performed as previously described. 29,33 mRNA was reversetranscribed using aminoallyl dUTP. The resulting cDNA from C57BL/6 tissue was labeled with Alexa Fluor 555 Reactive Dye (Invitrogen, Paisley, UK) and cDNA from SWR mouse tissue labeled with Alexa Fluor 647.…”

“…We used our cDNA arrays 29 to compare the expression of many known genes associated with iron metabolism in the liver, spleen, and duodenum of males of both mouse strains (Table 4). These cDNA arrays broadly contain those genes linked to iron and heme metabolism and were first reported by Muckenthaler.…”