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Regulation of neuroglobin, cytoglobin and myoglobin in … · Frank Gerlach1,2, Aaron Avivi3,...
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Expression analysis of Ngb mRNA and Protein Spalax constitutively expresses 1.8-2.7 fold more Ngb mRNA (realtime RT-PCR; *p<0.05) than rat under normoxic conditions and several fold more Ngb protein (Western Blot). Under hypoxia, Ngb mRNA is down-regulated by approximately 2-fold (realtime RT-PCR). Significant down-regulation occurs in all species ((*)p<0.1; *p<0.05; p<0.01). The most hypoxia resistant species S. galili (2n=52) does not yet react by down-regulation after 5h of 6% oxygen. Expression analysis of Cygb in different tissues Cygb is present in fibroblasts (FB) of the heart, kidney and colon. We also find Cygb staining in neuronal cells of the brain (AH and DG) and hepatic stellate cells of the liver. This cellular expression pattern is the same in Spalax and other mammals. Expression analysis of Cygb mRNA While Cygb expression levels are similar in rat and Spalax heart and liver, the brain of Spalax harbors near 3 times more Cygb mRNA than the rat brain. In Spalax but not rat, we observe a higher Cygb level in brain after 44h of 9%oxygen. Cygb is also up-regulated in heart of rat and Spalax, but in Spalax to a much higher extent. Expression analysis of Mb mRNA Mb is constitutively more highly expressed (27-43 fold) in neck muscle of both Spalax species as compared to rat. Under short, severe hypoxia (5h, 6% oxygen) the expression rises 2.7-fold in rat. Mb expression does not change in Spalax under these conditions. Mild, long-time hypoxia leads to a down- regulation of Mb mRNA. Introduction Neuroglobin (Ngb) and Cytoglobin (Cygb) are two recently discovered O 2 binding proteins [1,2]. Ngb is primarily expressed in neurons of the CNS and PNS. Cygb is found in fibroblasts and related cells and in distinct neurons. The physiological function of both globins is discussed in terms of O supply, ROS 2 scavenging, NO detoxification and other mechanisms [3]. Here we investigate the expression of Ngb, Cygb and Myoglobin (Mb) in the blind mole rat Spalax ehrenbergi and rat. We compare the expression levels at normoxia and under different hypoxic conditions. Among mammals, Spalax is a unique model of hypoxia tolerance [4,5]. It lives in underground burrows and survives prolonged times of severe hypoxia (11h of 3% oxygen) without neurological damage. In Israel, there are four different chromosomal species, which differ slightly in their hypoxia tolerance. S. galili (2n=52), which lives in flooding areas, is the most tolerant species, whereas S. judaei (2n=60) is slightly more sensitive towards oxygen deprivation. In contrast, rats are near death after 4h of 3% oxygen [6], representing the majority of non-hypoxia-adapted mammals. 1,2 3 3 1 2 Frank Gerlach , Aaron Avivi , Eviatar Nevo , Thorsten Burmester , Thomas Hankeln 1 , 2 Institute of Molecular Genetics, Johannes Gutenberg-University Mainz, 55099 Mainz, Germany, and 3 Institute of Evolution, Haifa University, Haifa 31905, Israel. Biocenter Grindel, University of Hamburg, 20146 Hamburg, Germany Regulation of neuroglobin, cytoglobin and myoglobin in a hypoxia- tolerant mammal, the subterranean mole rat Spalax ehrenbergi 27x 1.6x 27x 1.6x HSC HC FB Results Expression analysis of Ngb protein in Spalax (2n=54) brain tissue Ngb expression is restricted to neuronal cells, e.g. in the hippocampal area including Ammon´s horn (AH) and dentate gyrus (DG) (see right). We observe a typical cytoplasmic immunostaining signal including cellular processes. No differences between Spalax and Rattus were observed in Ngb protein or mRNA expression patterns. a -Cygb 1:100 a -Cygb 1:100 a -Cygb 1:100 a -Cygb 1:100 a -Cygb 1:100 a -Cygb 1:100 a -Cygb 1:100 a -Cygb 1:100 neg. control neg. control FB lumen FB kidney glomerulus unspecific erythrocyte staining FB ductus efferens brain liver heart colon kidney testes brain a -Ngb 1:50 a -Ngb 1:50 a -Ngb 1:50 DG AH AH DG 24 kDa 17 kDa 11 kDa Rat S52 S60 Rat S52 S60 Rat S52 S60 Indiv# 1 1 1 2 2 2 3 3 3 Discussion The data suggest that Ngb and Mb contribute to a pre-adaptation of Spalax towards a lack of O by sharing several fold stronger expression under normoxia. After extended periods of 2 hypoxia, both globins are down-regulated at the mRNA level in Spalax and rat, showing that an increase in O binding capacity is possibly not advantageous in these mammals. The 2 largely parallel pattern of gene regulation of Ngb and Mb suggests similar cellular functions, possibly in O supply and/or the detoxification of NO. 2 Cygb shows an augmented normoxic expression in Spalax vs. rat only in brain tissue, but not in heart and liver. Hypoxia specifically induces Cygb in heart and liver of both mammals, with the most pronounced response (12x up) in Spalax heart. The data indicate that Cygb possibly fulfils different roles in fibroblast-like cells than in neurons. Literature [1] Burmester T., Weich B., Reinhardt S., and Hankeln T. Nature 407 (2000), 520-523. [2] Burmester T., Ebner B., Weich B., and Hankeln T. Mol. Biol. Evol. 19 (2002), 416-421. [3] Hankeln et al. J. Inorg. Biochem. 99 (2005), 110-119. [4] Nevo, E. Oxford Science Publications (1999). [5] Nevo, E., Ivanitskaya, E., and Beiles, A. Backhuys Publishers, Leiden (2001). [6] Avivi A., Resnick M. B., Nevo E., Joel A., and Levy A. P. FEBS Letters 452 (1999), 133-140. Western Blot
Transcript of Regulation of neuroglobin, cytoglobin and myoglobin in … · Frank Gerlach1,2, Aaron Avivi3,...
Expression analysis of Ngb mRNA and Protein
Spalax constitutively expresses 1.8-2.7 fold more Ngb mRNA (realtime RT-PCR; *p<0.05) than rat under normoxic conditions and several fold more Ngb protein (Western Blot).
Under hypoxia, Ngb mRNA is down-regulated by approximately 2-fold (realtime RT-PCR). Significant down-regulation occurs in all species ((*)p<0.1; *p<0.05; p<0.01). The most hypoxia resistant species S. galili (2n=52) does not yet react by down-regulation after 5h of 6% oxygen.
Expression analysis of Cygb in different tissues
Cygb is present in fibroblasts (FB) of the heart, kidney and colon. We also find Cygb staining in neuronal cells of the brain (AH and DG) and hepatic stellate cells of the liver. This cellular expression pattern is the same in Spalax and other mammals.
Expression analysis of Cygb mRNA
While Cygb expression levels are similar in rat and Spalax heart and liver, the brain of Spalax harbors near 3 times more Cygb mRNA than the rat brain.
In Spalax but not rat, we observe a higher Cygb level in brain after 44h of 9%oxygen. Cygb is also up-regulated in heart of rat and Spalax, but in Spalax to a much higher extent.
Expression analysis of Mb mRNA
Mb is constitutively more highly expressed (27-43 fold) in neck muscle of both S p a l a x s p e c i e s a s compared to rat. Under short, severe hypoxia (5h, 6% oxygen) the expression rises 2.7-fold in rat. Mb expression does not change in Spalax under these conditions. Mild, long-time hypoxia leads to a down-regulation of Mb mRNA.
Introduction
Neuroglobin (Ngb) and Cytoglobin (Cygb) are two recent ly d iscovered O 2
binding proteins [1,2]. Ngb is pr imar i ly expressed in neurons of the CNS and PNS. Cygb is found in fibroblasts and related cells and in d i s t i n c t n e u r o n s . T h e physiological function of both globins is discussed in terms o f O s u p p l y , R O S 2
scavenging, NO detoxification and other mechanisms [3]. Here we investigate the expression of Ngb, Cygb and Myoglobin (Mb) in the blind mole rat Spalax ehrenbergi and rat. We compare the expression levels at normoxia and under different hypoxic conditions. Among mammals, Spalax is a unique model of hypoxia tolerance [4,5]. It lives in underground burrows and
survives prolonged times of severe hypoxia (11h of 3% oxygen) without neurological damage. In Israel, there are four different chromosomal species, which differ slightly in their hypoxia tolerance. S. galili (2n=52), which lives in flooding areas, is the most tolerant species, whereas S. judaei (2n=60) is slightly more sensitive towards oxygen deprivation. In contrast, rats are near death after 4h of 3% oxygen [6], representing the majority of non-hypoxia-adapted mammals.
1,2 3 3 1 2Frank Gerlach , Aaron Avivi , Eviatar Nevo , Thorsten Burmester , Thomas Hankeln
1 , 2Institute of Molecular Genetics, Johannes Gutenberg-University Mainz, 55099 Mainz, Germany, and
3Institute of Evolution, Haifa University, Haifa 31905, Israel.
Biocenter Grindel, University of Hamburg, 20146 Hamburg, Germany
Regulation of neuroglobin, cytoglobin and myoglobin in a hypoxia-tolerant mammal, the subterranean mole rat Spalax ehrenbergi
27x
1.6x
27x
1.6x
HSC
HC
FB
Results
Expression analysis of Ngb protein in Spalax (2n=54) brain t issue
Ngb expression is restricted to neuronal cells, e.g. in the hippocampal area including Ammon´s horn (AH) and dentate gyrus (DG) (see right). We observe a typical cytoplasmic immunostaining signal including cellular processes. No differences between Spalax and Rattus were observed in Ngb protein or mRNA expression patterns.
a-Cygb 1:100 a-Cygb 1:100
a-Cygb 1:100
a-Cygb 1:100a-Cygb 1:100 a-Cygb 1:100
a-Cygb 1:100
a-Cygb 1:100
neg. control
neg. control
FB
lumen
FB
kidneyglomerulus
unspecificerythrocyte staining
FB
ductusefferens
brain
liver
heart colon
kidney
testes
brain
a-Ngb 1:50 a-Ngb 1:50 a-Ngb 1:50
DGAH
AH
DG
24 kDa17 kDa
11 kDaRat S52 S60 Rat S52 S60 Rat S52 S60
Indiv# 1 1 1 2 2 2 3 3 3
Discussion
The data suggest that Ngb and Mb contribute to a pre-adaptation of Spalax towards a lack of O by sharing several fold stronger expression under normoxia. After extended periods of 2
hypoxia, both globins are down-regulated at the mRNA level in Spalax and rat, showing that an increase in O binding capacity is possibly not advantageous in these mammals. The 2
largely parallel pattern of gene regulation of Ngb and Mb suggests similar cellular functions, possibly in O supply and/or the detoxification of NO.2
Cygb shows an augmented normoxic expression in Spalax vs. rat only in brain tissue, but not in heart and liver. Hypoxia specifically induces Cygb in heart and liver of both mammals, with the most pronounced response (12x up) in Spalax heart. The data indicate that Cygb possibly fulfils different roles in fibroblast-like cells than in neurons.
Literature[1] Burmester T., Weich B., Reinhardt S., and Hankeln T. Nature 407 (2000), 520-523.[2] Burmester T., Ebner B., Weich B., and Hankeln T. Mol. Biol. Evol. 19 (2002), 416-421.[3] Hankeln et al. J. Inorg. Biochem. 99 (2005), 110-119.[4] Nevo, E. Oxford Science Publications (1999).[5] Nevo, E., Ivanitskaya, E., and Beiles, A. Backhuys Publishers, Leiden (2001).[6] Avivi A., Resnick M. B., Nevo E., Joel A., and Levy A. P. FEBS Letters 452 (1999), 133-140.
Western Blot
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