Activation of mesangial cell MAPK in response to homocysteine

Alistair J. Ingram, Joan C. Krepinsky, Leighton R James, Richard C. Austin, Damu Tang, Anne Marie Salapatek, Kerri Thai, James W. Scholey

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Background. Alteration in mesangial cell function is central to the progression of glomerular disease in numerous models of chronic renal failure (CRF). Animal models of chronic glomerular disease are characterized by mesangial cell proliferation and elaboration of extracellular matrix protein (ECM), resulting in glomerulosclerosis. Elevated plasma levels of homocysteine (Hcy) are seen in both animal models and humans with CRF, and have been proposed to contribute to the high prevalence of vascular disease in this group. Some of the pathogenetic effects of Hcy are thought to be mediated via the induction of endoplasmic reticulum stress. Thus, Hcy effects on mesangial cells could contribute to the progression of CRF. Previous work has shown Hcy- mediated induction of Erk mitogen-activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). Erk induces increases in activator protein-1 (AP-1) transcription factor activity which may augment mesangial cell proliferation and ECM protein production. Consequently, we studied the effect of Hcy on mesangial cell Erk signaling. Methods. Mesangial cells were exposed to Hcy after 24 hours of serum starvation and Erk activity assessed. Nuclear translocation of phospho-Erk was visualized by confocal microscopy. AP-1 nuclear protein binding was measured in response to Hcy by mobility shift assay. Hcy-induced mesangial cell calcium flux was measured in Fura-2 loaded cells. Mesangial cell DNA synthesis in response to Hcy was assessed by [3H]-thymidine incorporation and proliferation by Western blotting for proliferating cell nuclear antigen (PCNA). Expression of endoplasmic reticulum stress response genes were determined by Northern and Western analysis. Results. Hcy led to an increase in Erk activity that was maximal at 50 μmol/L and 20 minutes of treatment. Subsequent experiments used this concentration and time point. Erk activity in response to Hcy was insensitive to n-acetylcysteine and catalase, indicating oxidative stress did not play a role. However, Hcy 50 μmol/L induced a brief increase in intracellular mesangial cell calcium within 5 minutes, and the calcium ionophores A23187 and ionomycin increased Erk activity while chelation of intracellular calcium with BAPTA-AM abrogated the Erk response to Hcy. Confocal microscopy of activated Erk nuclear translocation mirrored these results as did mesangial cell nuclear protein binding to AP-1 consensus sequences. Hcy- induced increases in thymidine incorporation and PCNA expression at 24 hours were Erk dependent. The expression of endoplasmic reticulum stress response genes was significantly elevated by Hcy in an Erk-dependent manner. Conclusion. Hcy increases Erk activity in mesangial cells via a calcium-dependent mechanism, resulting in increased AP-1 nuclear protein binding, cell DNA synthesis and proliferation and induction of endoplasmic reticulum stress. These observations suggest potential mechanisms by which Hcy may contribute to progressive glomerular injury.

Original languageEnglish (US)
Pages (from-to)733-745
Number of pages13
JournalKidney International
Volume66
Issue number2
DOIs
StatePublished - Jan 1 2004
Externally publishedYes

Fingerprint

Mesangial Cells
Homocysteine
Mitogen-Activated Protein Kinases
Endoplasmic Reticulum Stress
Transcription Factor AP-1
Nuclear Proteins
Replication Protein C
Protein Binding
Chronic Kidney Failure
Calcium
Extracellular Matrix Proteins
Proliferating Cell Nuclear Antigen
Confocal Microscopy
Thymidine
Animal Models
Cell Proliferation
Ionomycin
Calcium Ionophores
Fura-2
DNA

Keywords

  • Homocysteine
  • Mesangial cell
  • Mitogen-activated protein kinase
  • Signaling

ASJC Scopus subject areas

  • Nephrology

Cite this

Ingram, A. J., Krepinsky, J. C., James, L. R., Austin, R. C., Tang, D., Salapatek, A. M., ... Scholey, J. W. (2004). Activation of mesangial cell MAPK in response to homocysteine. Kidney International, 66(2), 733-745. https://doi.org/10.1111/j.1523-1755.2004.00795.x

Activation of mesangial cell MAPK in response to homocysteine. / Ingram, Alistair J.; Krepinsky, Joan C.; James, Leighton R; Austin, Richard C.; Tang, Damu; Salapatek, Anne Marie; Thai, Kerri; Scholey, James W.

In: Kidney International, Vol. 66, No. 2, 01.01.2004, p. 733-745.

Research output: Contribution to journalArticle

Ingram, AJ, Krepinsky, JC, James, LR, Austin, RC, Tang, D, Salapatek, AM, Thai, K & Scholey, JW 2004, 'Activation of mesangial cell MAPK in response to homocysteine', Kidney International, vol. 66, no. 2, pp. 733-745. https://doi.org/10.1111/j.1523-1755.2004.00795.x
Ingram AJ, Krepinsky JC, James LR, Austin RC, Tang D, Salapatek AM et al. Activation of mesangial cell MAPK in response to homocysteine. Kidney International. 2004 Jan 1;66(2):733-745. https://doi.org/10.1111/j.1523-1755.2004.00795.x
Ingram, Alistair J. ; Krepinsky, Joan C. ; James, Leighton R ; Austin, Richard C. ; Tang, Damu ; Salapatek, Anne Marie ; Thai, Kerri ; Scholey, James W. / Activation of mesangial cell MAPK in response to homocysteine. In: Kidney International. 2004 ; Vol. 66, No. 2. pp. 733-745.
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AU - Ingram, Alistair J.

AU - Krepinsky, Joan C.

AU - James, Leighton R

AU - Austin, Richard C.

AU - Tang, Damu

AU - Salapatek, Anne Marie

AU - Thai, Kerri

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N2 - Background. Alteration in mesangial cell function is central to the progression of glomerular disease in numerous models of chronic renal failure (CRF). Animal models of chronic glomerular disease are characterized by mesangial cell proliferation and elaboration of extracellular matrix protein (ECM), resulting in glomerulosclerosis. Elevated plasma levels of homocysteine (Hcy) are seen in both animal models and humans with CRF, and have been proposed to contribute to the high prevalence of vascular disease in this group. Some of the pathogenetic effects of Hcy are thought to be mediated via the induction of endoplasmic reticulum stress. Thus, Hcy effects on mesangial cells could contribute to the progression of CRF. Previous work has shown Hcy- mediated induction of Erk mitogen-activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). Erk induces increases in activator protein-1 (AP-1) transcription factor activity which may augment mesangial cell proliferation and ECM protein production. Consequently, we studied the effect of Hcy on mesangial cell Erk signaling. Methods. Mesangial cells were exposed to Hcy after 24 hours of serum starvation and Erk activity assessed. Nuclear translocation of phospho-Erk was visualized by confocal microscopy. AP-1 nuclear protein binding was measured in response to Hcy by mobility shift assay. Hcy-induced mesangial cell calcium flux was measured in Fura-2 loaded cells. Mesangial cell DNA synthesis in response to Hcy was assessed by [3H]-thymidine incorporation and proliferation by Western blotting for proliferating cell nuclear antigen (PCNA). Expression of endoplasmic reticulum stress response genes were determined by Northern and Western analysis. Results. Hcy led to an increase in Erk activity that was maximal at 50 μmol/L and 20 minutes of treatment. Subsequent experiments used this concentration and time point. Erk activity in response to Hcy was insensitive to n-acetylcysteine and catalase, indicating oxidative stress did not play a role. However, Hcy 50 μmol/L induced a brief increase in intracellular mesangial cell calcium within 5 minutes, and the calcium ionophores A23187 and ionomycin increased Erk activity while chelation of intracellular calcium with BAPTA-AM abrogated the Erk response to Hcy. Confocal microscopy of activated Erk nuclear translocation mirrored these results as did mesangial cell nuclear protein binding to AP-1 consensus sequences. Hcy- induced increases in thymidine incorporation and PCNA expression at 24 hours were Erk dependent. The expression of endoplasmic reticulum stress response genes was significantly elevated by Hcy in an Erk-dependent manner. Conclusion. Hcy increases Erk activity in mesangial cells via a calcium-dependent mechanism, resulting in increased AP-1 nuclear protein binding, cell DNA synthesis and proliferation and induction of endoplasmic reticulum stress. These observations suggest potential mechanisms by which Hcy may contribute to progressive glomerular injury.

AB - Background. Alteration in mesangial cell function is central to the progression of glomerular disease in numerous models of chronic renal failure (CRF). Animal models of chronic glomerular disease are characterized by mesangial cell proliferation and elaboration of extracellular matrix protein (ECM), resulting in glomerulosclerosis. Elevated plasma levels of homocysteine (Hcy) are seen in both animal models and humans with CRF, and have been proposed to contribute to the high prevalence of vascular disease in this group. Some of the pathogenetic effects of Hcy are thought to be mediated via the induction of endoplasmic reticulum stress. Thus, Hcy effects on mesangial cells could contribute to the progression of CRF. Previous work has shown Hcy- mediated induction of Erk mitogen-activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). Erk induces increases in activator protein-1 (AP-1) transcription factor activity which may augment mesangial cell proliferation and ECM protein production. Consequently, we studied the effect of Hcy on mesangial cell Erk signaling. Methods. Mesangial cells were exposed to Hcy after 24 hours of serum starvation and Erk activity assessed. Nuclear translocation of phospho-Erk was visualized by confocal microscopy. AP-1 nuclear protein binding was measured in response to Hcy by mobility shift assay. Hcy-induced mesangial cell calcium flux was measured in Fura-2 loaded cells. Mesangial cell DNA synthesis in response to Hcy was assessed by [3H]-thymidine incorporation and proliferation by Western blotting for proliferating cell nuclear antigen (PCNA). Expression of endoplasmic reticulum stress response genes were determined by Northern and Western analysis. Results. Hcy led to an increase in Erk activity that was maximal at 50 μmol/L and 20 minutes of treatment. Subsequent experiments used this concentration and time point. Erk activity in response to Hcy was insensitive to n-acetylcysteine and catalase, indicating oxidative stress did not play a role. However, Hcy 50 μmol/L induced a brief increase in intracellular mesangial cell calcium within 5 minutes, and the calcium ionophores A23187 and ionomycin increased Erk activity while chelation of intracellular calcium with BAPTA-AM abrogated the Erk response to Hcy. Confocal microscopy of activated Erk nuclear translocation mirrored these results as did mesangial cell nuclear protein binding to AP-1 consensus sequences. Hcy- induced increases in thymidine incorporation and PCNA expression at 24 hours were Erk dependent. The expression of endoplasmic reticulum stress response genes was significantly elevated by Hcy in an Erk-dependent manner. Conclusion. Hcy increases Erk activity in mesangial cells via a calcium-dependent mechanism, resulting in increased AP-1 nuclear protein binding, cell DNA synthesis and proliferation and induction of endoplasmic reticulum stress. These observations suggest potential mechanisms by which Hcy may contribute to progressive glomerular injury.

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KW - Mitogen-activated protein kinase

KW - Signaling

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