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Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1.

Mitchell RM, Lee SY, Randazzo WT, Simmons Z, Connor JR - J Neuroinflammation (2009)

Bottom Line: The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways.We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status.Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: George M Leader Family Laboratory, Department of Neurosurgery, Pennsylvania State University College of Medicine/Milton S Hershey Medical Center, Hershey, PA 17033, USA. rmm311@psu.edu

ABSTRACT

Background: Polymorphisms in the MHC class 1-like gene known as HFE have been proposed as genetic modifiers of neurodegenerative diseases that include neuroinflammation as part of the disease process. Variants of HFE are relatively common in the general population and are most commonly associated with iron overload, but can promote subclinical cellular iron loading even in the absence of clinically identified disease. The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways. We tested the hypothesis that the two most common HFE variants, H63D and C282Y, would affect cellular secretion of cytokines and trophic factors.

Methods: We screened a panel of cytokines and trophic factors using a multiplexed immunoassay in human neuroblastoma SH-SY5Y cells expressing different variants of HFE. The influence of cellular iron secretion on the potent chemokine monocyte chemoattractant protein-1 (MCP-1) was assessed using ferric ammonium citrate and the iron chelator, desferroxamine. Additionally, an antioxidant, Trolox, and an anti-inflammatory, minocycline, were tested for their effects on MCP-1 secretion in the presence of HFE variants.

Results: Expression of the HFE variants altered the labile iron pool in SH-SY5Y cells. Of the panel of cytokines and trophic factors analyzed, only the release of MCP-1 was affected by the HFE variants. We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status. A potential direct effect of HFE is considered because, despite having similar levels of intracellular iron, the association between HFE genotype and MCP-1 expression was different for the H63D and C282Y HFE variants. Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.

Conclusion: Our results demonstrate that HFE polymorphisms influence the synthesis and release of MCP-1. The mechanism of action involves cellular iron status but it appears there could be additional influences such as ER stress. Finally, these data demonstrate a pharmacogenetic effect of HFE polymorphisms on the ability of minocycline to inhibit MCP-1 secretion.

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MCP-1 mRNA expression and nuclear NF-κB. (A) RNA was isolated from five separate cultures of differentiated SH-SY5Y cells and used for quantitative RT-PCR. Expression of MCP-1 mRNA was normalized to the expression of GAPDH mRNA. MCP-1 mRNA expression was 3.0-, 3.2-, and 4.1-fold higher in vector-transfected, H63D HFE, and C282Y HFE cells, respectively, compared to cells expressing Wt HFE. (B) Nuclear fractions were isolated from four independent cultures of stably-transfected SH-SY5Y cell lines. Nuclear content of NF-κB (p50) was assessed by colorimetric assay and reported as optical density per milligram of nuclear protein. Results are expressed as mean ± SEM. No differences in NF-κB (p50) were found between any of the SH-SY5Y cell lines. n = 4. * and ** represent p < 0.05 and p < 0.01, respectively, compared to Wt HFE cells.
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Figure 3: MCP-1 mRNA expression and nuclear NF-κB. (A) RNA was isolated from five separate cultures of differentiated SH-SY5Y cells and used for quantitative RT-PCR. Expression of MCP-1 mRNA was normalized to the expression of GAPDH mRNA. MCP-1 mRNA expression was 3.0-, 3.2-, and 4.1-fold higher in vector-transfected, H63D HFE, and C282Y HFE cells, respectively, compared to cells expressing Wt HFE. (B) Nuclear fractions were isolated from four independent cultures of stably-transfected SH-SY5Y cell lines. Nuclear content of NF-κB (p50) was assessed by colorimetric assay and reported as optical density per milligram of nuclear protein. Results are expressed as mean ± SEM. No differences in NF-κB (p50) were found between any of the SH-SY5Y cell lines. n = 4. * and ** represent p < 0.05 and p < 0.01, respectively, compared to Wt HFE cells.

Mentions: The mechanism by which HFE variants may differentially regulate synthesis of MCP-1 was explored at the transcription level. RNA was isolated from each SH-SY5Y cell line for measurement of MCP-1 mRNA expression. After normalizing to the expression of GAPDH mRNA, MCP-1 mRNA expression was 3.0- (p < 0.05), 3.2- (p < 0.05), and 4.1-fold higher (p < 0.01) in vector-transfected, H63D HFE, and C282Y HFE cells, respectively, compared to cells expressing Wt HFE (Figure 3A). Between cells expressing Wt HFE and those expressing H63D HFE or even the vector alone cells, MCP-1 mRNA expression followed the pattern of MCP-1 secretion. Despite less secretion of MCP-1 associated with the C282Y HFE variant compared to H63D HFE, however, MCP-1 mRNA expression was not significantly different between the two cell lines. These data suggest MCP-1 is regulated at both the transcriptional and post-transcriptional levels and that the mutant proteins or iron status can affect the regulation.


Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1.

Mitchell RM, Lee SY, Randazzo WT, Simmons Z, Connor JR - J Neuroinflammation (2009)

MCP-1 mRNA expression and nuclear NF-κB. (A) RNA was isolated from five separate cultures of differentiated SH-SY5Y cells and used for quantitative RT-PCR. Expression of MCP-1 mRNA was normalized to the expression of GAPDH mRNA. MCP-1 mRNA expression was 3.0-, 3.2-, and 4.1-fold higher in vector-transfected, H63D HFE, and C282Y HFE cells, respectively, compared to cells expressing Wt HFE. (B) Nuclear fractions were isolated from four independent cultures of stably-transfected SH-SY5Y cell lines. Nuclear content of NF-κB (p50) was assessed by colorimetric assay and reported as optical density per milligram of nuclear protein. Results are expressed as mean ± SEM. No differences in NF-κB (p50) were found between any of the SH-SY5Y cell lines. n = 4. * and ** represent p < 0.05 and p < 0.01, respectively, compared to Wt HFE cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2656486&req=5

Figure 3: MCP-1 mRNA expression and nuclear NF-κB. (A) RNA was isolated from five separate cultures of differentiated SH-SY5Y cells and used for quantitative RT-PCR. Expression of MCP-1 mRNA was normalized to the expression of GAPDH mRNA. MCP-1 mRNA expression was 3.0-, 3.2-, and 4.1-fold higher in vector-transfected, H63D HFE, and C282Y HFE cells, respectively, compared to cells expressing Wt HFE. (B) Nuclear fractions were isolated from four independent cultures of stably-transfected SH-SY5Y cell lines. Nuclear content of NF-κB (p50) was assessed by colorimetric assay and reported as optical density per milligram of nuclear protein. Results are expressed as mean ± SEM. No differences in NF-κB (p50) were found between any of the SH-SY5Y cell lines. n = 4. * and ** represent p < 0.05 and p < 0.01, respectively, compared to Wt HFE cells.
Mentions: The mechanism by which HFE variants may differentially regulate synthesis of MCP-1 was explored at the transcription level. RNA was isolated from each SH-SY5Y cell line for measurement of MCP-1 mRNA expression. After normalizing to the expression of GAPDH mRNA, MCP-1 mRNA expression was 3.0- (p < 0.05), 3.2- (p < 0.05), and 4.1-fold higher (p < 0.01) in vector-transfected, H63D HFE, and C282Y HFE cells, respectively, compared to cells expressing Wt HFE (Figure 3A). Between cells expressing Wt HFE and those expressing H63D HFE or even the vector alone cells, MCP-1 mRNA expression followed the pattern of MCP-1 secretion. Despite less secretion of MCP-1 associated with the C282Y HFE variant compared to H63D HFE, however, MCP-1 mRNA expression was not significantly different between the two cell lines. These data suggest MCP-1 is regulated at both the transcriptional and post-transcriptional levels and that the mutant proteins or iron status can affect the regulation.

Bottom Line: The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways.We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status.Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: George M Leader Family Laboratory, Department of Neurosurgery, Pennsylvania State University College of Medicine/Milton S Hershey Medical Center, Hershey, PA 17033, USA. rmm311@psu.edu

ABSTRACT

Background: Polymorphisms in the MHC class 1-like gene known as HFE have been proposed as genetic modifiers of neurodegenerative diseases that include neuroinflammation as part of the disease process. Variants of HFE are relatively common in the general population and are most commonly associated with iron overload, but can promote subclinical cellular iron loading even in the absence of clinically identified disease. The effects of the variants as well as the resulting cellular iron dyshomeostasis potentially impact a number of disease-associated pathways. We tested the hypothesis that the two most common HFE variants, H63D and C282Y, would affect cellular secretion of cytokines and trophic factors.

Methods: We screened a panel of cytokines and trophic factors using a multiplexed immunoassay in human neuroblastoma SH-SY5Y cells expressing different variants of HFE. The influence of cellular iron secretion on the potent chemokine monocyte chemoattractant protein-1 (MCP-1) was assessed using ferric ammonium citrate and the iron chelator, desferroxamine. Additionally, an antioxidant, Trolox, and an anti-inflammatory, minocycline, were tested for their effects on MCP-1 secretion in the presence of HFE variants.

Results: Expression of the HFE variants altered the labile iron pool in SH-SY5Y cells. Of the panel of cytokines and trophic factors analyzed, only the release of MCP-1 was affected by the HFE variants. We further examined the relationship between iron and MCP-1 and found MCP-1 secretion tightly associated with intracellular iron status. A potential direct effect of HFE is considered because, despite having similar levels of intracellular iron, the association between HFE genotype and MCP-1 expression was different for the H63D and C282Y HFE variants. Moreover, HFE genotype was a factor in the effect of minocycline, a multifaceted antibiotic used in treating a number of neurologic conditions associated with inflammation, on MCP-1 secretion.

Conclusion: Our results demonstrate that HFE polymorphisms influence the synthesis and release of MCP-1. The mechanism of action involves cellular iron status but it appears there could be additional influences such as ER stress. Finally, these data demonstrate a pharmacogenetic effect of HFE polymorphisms on the ability of minocycline to inhibit MCP-1 secretion.

Show MeSH
Related in: MedlinePlus