Fox MA et al. (2011), PEX11β induces peroxisomal gene expression and ...

XB-ART-43157

BMC Dev Biol 2011 Apr 28;11:24. doi: 10.1186/1471-213X-11-24.

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PEX11β induces peroxisomal gene expression and alters peroxisome number during early Xenopus laevis development.

Fox MA , Walsh LA , Nieuwesteeg M , Damjanovski S .

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BACKGROUND: Peroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species elimination have contributed much attention in understanding their origin and biogenesis. Many studies have shown that de novo peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in part regulated by proteins such as Pex11, which can facilitate the division of existing peroxisomes. Although de novo biogenesis and divisions are likely important mechanisms, the regulation of peroxisome numbers during embryonic development is poorly understood. Peroxisome number and function are particularly crucial in oviparous animals such as frogs where large embryonic yolk and fatty acid stores must be quickly metabolized, and resulting reactive oxygen species eliminated. Here we elucidate the role of Pex11β in regulating peroxisomal gene expression and number in Xenopus laevis embryogenesis. RESULTS: Microinjecting haemagglutinin (HA) tagged Pex11β in early embryos resulted in increased RNA levels for peroxisome related genes PMP70 and catalase at developmental stages 10 and 20, versus uninjected embryos. Catalase and PMP70 proteins were found in punctate structures at stage 20 in control embryos, whereas the injection of ectopic HA-Pex11β induced their earlier localization in punctate structures at stage 10. Furthermore, the peroxisomal marker GFP-SKL, which was found localized as peroxisome-like structures at stage 20, was similarly found at stage 10 when co-microinjected with HA-Pex11β. CONCLUSIONS: Overexpressed Pex11β altered peroxisomal gene levels and induced the early formation of peroxisomes-like structures during development, both of which demonstrate that Pex11β may be a key regulator of peroxisome number in early Xenopus embryos.

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Species referenced: Xenopus laevis
Genes referenced: abcd3 actl6a cat.1 pex11b pex3 pex5

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	Figure 3. Overexpression of HA-Pex11β in A6 cells increased peroxisome numbers. A, D, G are untransfected cells, while B, E, H have been transfected with HA-Pex11β. G and H are additionally transfected with GFP-SKL. Using identical imaging and photography parameters, indirect immunofluorescence using a catalase antibody revealed lower levels of immunofluorescence in untransfected cells (A) versus transfected (B) cells. Similarly, indirect immunofluorescence using a PMP70 antibody revealed lower levels of signal in untransfected cells (D) versus transfected (E). Direct fluorescence for GFP revealed a diffuse signal from GFP in HA-Pex11β untransfected cells, (G) versus the presence of punctate structures in HA-Pex11β transfected cells (H). All images were captured using identical fluorescent settings. The relative fluorescence intensity in 10 regions of twenty randomly imaged cells was quantified using Northern Eclipse software. Graphs on the right represent the average fluorescence intensity of untransfected versus HA-Pex11β transfected cells. Values presented are the means ± SE. Significance at P < 0.05 was determined using Student's t-test, n = 25.
	Figure 6. Microinjecting HA-Pex11β RNA increased PMP70 immunofluorescence levels during X. laevis embryogenesis. Both control (A and B) and HA-Pex11β injected (C and D) embryos at developmental stages 10 (A and C) and 20 (B and D), were fixed then sectioned for immunohistochemical analysis in somites for PMP70. At stage 10 PMP70 protein is undetected in somitic mesoderm under control conditions (A), whereas following microinjection of HA-Pex11β PMP70 protein is detectable in punctate structures (C). At stage 20, PMP70 protein was detected in both control and following microinjecting HA-Pex11β (B and D). An HA antibody was also used to confirm the ectopic presence of HA-Pex11β. DAPI (blue), PMP70 (green), HA-Pex11β (red), colocalization of HA-Pex11β and catalase (yellow). Images were taken at 60magnification.
	Figure 7. Microinjecting HA-Pex11β RNA increased catalase immunofluorescence levels during X. laevis embryogenesis. Both control (A and B) and HA-Pex11β injected (C and D) embryos at developmental stages 10 (A and C) and 20 (B and D), were fixed then sectioned for immunohistochemical analysis in somites for catalase. At stage 10 catalase protein is undetected in somitic mesoderm under control conditions (A), whereas following microinjection of HA-Pex11β catalase protein is detectable in punctate structures (C). At stage 20, PMP70 protein was detected in both control and following microinjecting HA-Pex11β (B and D). An HA antibody was also used to confirm the ectopic presence of HA-Pex11β. DAPI (blue), PMP70 (green), HA-Pex11β (red), colocalization of HA-Pex11β and PMP70 (yellow). Images were taken at 60magnification.
	Figure 8. Microinjecting HA-Pex11β RNA increased the number of peroxisome-like GFP-SKL structures during X. laevis embryogenesis. Both control (A and B) and HA-Pex11β injected (C and D) embryos at developmental stages 10 (A and C) and 20 (B and D), were fixed then sectioned for immunohistochemical analysis in somites for GFP-SKL. At stage 10, GFP fluorescence is present as a faint diffuse stain under control conditions (A), whereas following microinjection of HA-Pex11β punctate GFP structures could be seen at this early stage (C). At stage 20, punctate GFP structures were detected in both control and following microinjecting HA-Pex11β, where numbers were increased in the injected samples. An HA antibody was also used to confirm the ectopic presence of HA-Pex11β. DAPI (blue), GFP (green), HA-Pex11β (red), colocalization of HA-Pex11β and GFP-SKL (yellow). Images were taken at 60magnification.
	Figure 1. Overexpressing HA-Pex11β altered peroxisome related gene expression in Xenopus A6 cells. RT-PCR analysis of peroxisomal genes was performed before and after transfection of A6 cells with HA-Pex11β. Two days following transfection 250 ng of reverse-transcribed A6 cell RNAs from control and treatment samples (n = 3) were subject to PCR amplification using specific primers for the peroxisome related genes; Pex11β, PMP70, catalase, Pex5, Pex3, Pex1, PPARα, -δ, and -γ. The respective mRNA levels represent measures of mid-log phase RT-PCR product band intensities, relative to levels of EF1α. Genes whose levels were altered significantly, as assessed by an independent samples t-test, are denoted with an asterisk. Pex11β, PMP70, catalase, Pex5 and PPARα displayed elevated levels of expression following treatment, while PPARγ displayed reduced expression. P < 0.05, n = 3. Values presented are the means ± SE.
	Figure 2. Overexpression of HA-Pex11β in A6 cells increased catalase and PMP70 protein levels. Western blotting revealed elevated proteins levels of catalase and PMP70 following the transfections of HA-Pex11β, but not GFP, in three samples of A6 cells (A). An HA antibody confirmed the translation and presence of HA-Pex11β in transfected cells (right 3 lanes) versus GFP transfected control cells (left 3 lanes). Catalase and PMP70 antibodies also displayed altered band intensities of each respective protein in HA-Pex11β transfected cells (right 3 lanes) versus GFP transfected cells (left 3 lanes). Protein loading in each lane was confirmed via a β-actin antibody. The Western blot signals were digitized and data were quantified and analyzed to statistically compare protein levels (B). There was a significant increase in the levels of catalase and PMP70 following overexpression of Pex11β, while there was no difference in the levels of β-actin. Statistical relevance of discrepancies between groups (asterisks) was assessed by an independent samples t-test. P < 0.05, n = 3. Values presented are the means ± SE.
	Figure 4. Embryonic overexpression of HA-Pex11β elevated Pex3, catalase and PMP70 levels. The respective mRNA levels represent measures of mid-log phase RT-PCR product band intensities, relative to levels of EF1α. RT-PCR analysis during normal embryogenesis revealed that the levels of all genes examined, with the exception of Pex5, increased as development progressed. First, repeated measures ANOVAs were carried out entering all RNA levels at all 3 stages. When significant, paired samples t-tests were carried out between stage levels in control embryos. This would reveal significant changes in RNA levels of the genes examined during normal development. Significant changes in RNA levels of a gene between stages is represented by the double asterisk ** (P < 0.05). Expression increases with development and there are differences in the levels of Pex3 and PMP70 between all stages, 10vs20, 20vs30 and 10 versus 30. For catalase there are differences between stages 10vs20 and 10vs30 but NOT between 20 versus 30. There are no significant differences in RNA levels between the tested developmental stages for Pex11β or Pex5. As the means were correlated, MANOVAs were carried out on the mean RNA levels of the treatment and control groups at each stage for each gene. All MANOVAs displayed significant effects of condition (Wilks lambda, P < 0.05) and so univariate ANOVAs were carried out. Treatments that resulted in significantly higher levels of gene expression following treatment are represented by a single asterisk. Pex11β, catalase and PMP70 all displayed significant increases in RNA levels all stage 10, stage 20 and stage 30 following treatment. Pex3 displayed elevated expression at only stage 10 and 30, while Pex5 displayed differences only at stages 20 and 30. (Ps < 0.05). n = 3. Values presented are the means ± SE.
	Figure 5. Overexpression of HA-Pex11β did altered PPARα and γ, but not δ, gene expression during early X. laevis embryogenesis. The respective mRNA levels represent measures of mid-log phase RT-PCR product band intensities, relative to levels of EF1α. Using a similar approach and analysis as used in Figure 4, RT-PCR analysis of RNA isolated from control stage 10 embryos and HA-Pex11β injected embryos revealed significant changes in the expression of PPARα, and PPARγ, but not PPARδ. PPARα levels were elevated by treatment, while PPARγ levels were reduced. P < 0.05, n = 3. Values presented are the means ± SE.

References [+] :

Anand, Hydrogen peroxide induced oxidation of peroxisomal malate synthase and catalase. 2009, Pubmed