Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells

A. J. Ingram, Leighton R James, H. Ly, K. Thai, L. Cai, J. W. Scholey

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Background. In vivo, intraglomerular hypertension results in resident cell hypertrophy, proliferation and matrix protein production, leading to glomerulosclerosis. Mesangial cells (MCs) exposed to in vitro stretch also proliferate and produce matrix. We have shown activation of Jun N-terminal kinase/stress-activated protein kinase (SAPK) and p42/44 mitogen-activated protein kinase (MAPK) in stretched MCs and have also demonstrated that L-arginine decreases resident cell proliferation and protects against glomerulosclerosis in remnant kidney glomeruli, presumably by increasing nitric oxide (NO) production. Consequently, we studied whether NO could affect SAPK and p42/44 MAPK activation in stretched MCs. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 0 to 30 minutes of cyclic strain (60 cycles per minute) by computer-driven generation of vacuum of -27 kPa, inducing 28% elongation in the diameter of the surface. Control MCs were grown on coated, flexible-bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK and p42/44 MAPK were performed under these conditions. As maximal activation was at 10 minutes, with decay by 30 minutes, the effect of NO on kinase activation was studied at 0, 2, 5, and 10 minutes by preincubation with 70 μmol/L s-nitroso-n-acetylpenicillamine (SNAP; an NO donor) or 1 mmol/L 8-bromo cyclic guanosine monophosphate (8-bromo-cGMP). Downstream events in response to stretch and NO were studied at the time of maximal response (10 minutes) by examining nuclear translocation of SAPK with immunofluorescence microscopy and transcription factor activator protein-1 nuclear protein binding by gel mobility shift assay. The effect of kinase inhibition by NO donors on MC proliferation was studied by Western blotting for proliferating cell nuclear antigen (PCNA). Results. Cyclic MC stretch led to prompt SAPK and p42/44 MAPK activation, which was maximal at 10 minutes. Preincubation with either SNAP or 8-bromo-cGMP decreased this by 50 and 70%, respectively (N = 4), suggesting that the effect of NO was through cGMP generation. Nuclear translocation of both phosphorylated kinases was seen after 10 minutes of stretch and was largely prevented by 8-bromo-cGMP. Increased DNA binding of activator protein-1 proteins was observed in the nuclei of stretched MCs at 10 minutes by mobility shift assay (N = 4), which was also largely prevented by 8-bromo-cGMP. Stretch increased PCNA expression by MCs, and this was inhibited by 8-bromo-cGMP. Conclusions. Stretch-induced activation of SAPK and p42/44 MAPK in MCs can be inhibited by NO. The effect of NO is mediated by the generation of cGMP. These mechanisms may be responsible, at least in part, for the protective effect of NO in animal models of glomerular injury characterized by glomerular capillary hypertension.

Original languageEnglish (US)
Pages (from-to)1067-1077
Number of pages11
JournalKidney International
Volume58
Issue number3
DOIs
StatePublished - Jan 1 2000
Externally publishedYes

Fingerprint

Mesangial Cells
Mitogen-Activated Protein Kinases
Nitric Oxide
Heat-Shock Proteins
Mitogen-Activated Protein Kinase 1
Protein Kinases
Cyclic GMP
Phosphotransferases
Nitric Oxide Donors
Transcription Factor AP-1
Cell Proliferation
Proliferating Cell Nuclear Antigen
Electrophoretic Mobility Shift Assay
Replication Protein C
Western Blotting
Kidney Glomerulus
Hypertension
Proteins
DNA-Binding Proteins
Vacuum

Keywords

  • Cell hypertrophy
  • Glomerulosclerosis
  • Jun N-terminal kinase
  • Mechanical strain
  • Stress-activated protein kinase

ASJC Scopus subject areas

  • Nephrology

Cite this

Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells. / Ingram, A. J.; James, Leighton R; Ly, H.; Thai, K.; Cai, L.; Scholey, J. W.

In: Kidney International, Vol. 58, No. 3, 01.01.2000, p. 1067-1077.

Research output: Contribution to journalArticle

Ingram, A. J. ; James, Leighton R ; Ly, H. ; Thai, K. ; Cai, L. ; Scholey, J. W. / Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells. In: Kidney International. 2000 ; Vol. 58, No. 3. pp. 1067-1077.
@article{9dc3b960f6b849a4a0a1258ebe9d2d45,
title = "Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells",
abstract = "Background. In vivo, intraglomerular hypertension results in resident cell hypertrophy, proliferation and matrix protein production, leading to glomerulosclerosis. Mesangial cells (MCs) exposed to in vitro stretch also proliferate and produce matrix. We have shown activation of Jun N-terminal kinase/stress-activated protein kinase (SAPK) and p42/44 mitogen-activated protein kinase (MAPK) in stretched MCs and have also demonstrated that L-arginine decreases resident cell proliferation and protects against glomerulosclerosis in remnant kidney glomeruli, presumably by increasing nitric oxide (NO) production. Consequently, we studied whether NO could affect SAPK and p42/44 MAPK activation in stretched MCs. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 0 to 30 minutes of cyclic strain (60 cycles per minute) by computer-driven generation of vacuum of -27 kPa, inducing 28{\%} elongation in the diameter of the surface. Control MCs were grown on coated, flexible-bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK and p42/44 MAPK were performed under these conditions. As maximal activation was at 10 minutes, with decay by 30 minutes, the effect of NO on kinase activation was studied at 0, 2, 5, and 10 minutes by preincubation with 70 μmol/L s-nitroso-n-acetylpenicillamine (SNAP; an NO donor) or 1 mmol/L 8-bromo cyclic guanosine monophosphate (8-bromo-cGMP). Downstream events in response to stretch and NO were studied at the time of maximal response (10 minutes) by examining nuclear translocation of SAPK with immunofluorescence microscopy and transcription factor activator protein-1 nuclear protein binding by gel mobility shift assay. The effect of kinase inhibition by NO donors on MC proliferation was studied by Western blotting for proliferating cell nuclear antigen (PCNA). Results. Cyclic MC stretch led to prompt SAPK and p42/44 MAPK activation, which was maximal at 10 minutes. Preincubation with either SNAP or 8-bromo-cGMP decreased this by 50 and 70{\%}, respectively (N = 4), suggesting that the effect of NO was through cGMP generation. Nuclear translocation of both phosphorylated kinases was seen after 10 minutes of stretch and was largely prevented by 8-bromo-cGMP. Increased DNA binding of activator protein-1 proteins was observed in the nuclei of stretched MCs at 10 minutes by mobility shift assay (N = 4), which was also largely prevented by 8-bromo-cGMP. Stretch increased PCNA expression by MCs, and this was inhibited by 8-bromo-cGMP. Conclusions. Stretch-induced activation of SAPK and p42/44 MAPK in MCs can be inhibited by NO. The effect of NO is mediated by the generation of cGMP. These mechanisms may be responsible, at least in part, for the protective effect of NO in animal models of glomerular injury characterized by glomerular capillary hypertension.",
keywords = "Cell hypertrophy, Glomerulosclerosis, Jun N-terminal kinase, Mechanical strain, Stress-activated protein kinase",
author = "Ingram, {A. J.} and James, {Leighton R} and H. Ly and K. Thai and L. Cai and Scholey, {J. W.}",
year = "2000",
month = "1",
day = "1",
doi = "10.1046/j.1523-1755.2000.00264.x",
language = "English (US)",
volume = "58",
pages = "1067--1077",
journal = "Kidney International",
issn = "0085-2538",
publisher = "Nature Publishing Group",
number = "3",

}

TY - JOUR

T1 - Nitric oxide modulates stretch activation of mitogen-activated protein kinases in mesangial cells

AU - Ingram, A. J.

AU - James, Leighton R

AU - Ly, H.

AU - Thai, K.

AU - Cai, L.

AU - Scholey, J. W.

PY - 2000/1/1

Y1 - 2000/1/1

N2 - Background. In vivo, intraglomerular hypertension results in resident cell hypertrophy, proliferation and matrix protein production, leading to glomerulosclerosis. Mesangial cells (MCs) exposed to in vitro stretch also proliferate and produce matrix. We have shown activation of Jun N-terminal kinase/stress-activated protein kinase (SAPK) and p42/44 mitogen-activated protein kinase (MAPK) in stretched MCs and have also demonstrated that L-arginine decreases resident cell proliferation and protects against glomerulosclerosis in remnant kidney glomeruli, presumably by increasing nitric oxide (NO) production. Consequently, we studied whether NO could affect SAPK and p42/44 MAPK activation in stretched MCs. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 0 to 30 minutes of cyclic strain (60 cycles per minute) by computer-driven generation of vacuum of -27 kPa, inducing 28% elongation in the diameter of the surface. Control MCs were grown on coated, flexible-bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK and p42/44 MAPK were performed under these conditions. As maximal activation was at 10 minutes, with decay by 30 minutes, the effect of NO on kinase activation was studied at 0, 2, 5, and 10 minutes by preincubation with 70 μmol/L s-nitroso-n-acetylpenicillamine (SNAP; an NO donor) or 1 mmol/L 8-bromo cyclic guanosine monophosphate (8-bromo-cGMP). Downstream events in response to stretch and NO were studied at the time of maximal response (10 minutes) by examining nuclear translocation of SAPK with immunofluorescence microscopy and transcription factor activator protein-1 nuclear protein binding by gel mobility shift assay. The effect of kinase inhibition by NO donors on MC proliferation was studied by Western blotting for proliferating cell nuclear antigen (PCNA). Results. Cyclic MC stretch led to prompt SAPK and p42/44 MAPK activation, which was maximal at 10 minutes. Preincubation with either SNAP or 8-bromo-cGMP decreased this by 50 and 70%, respectively (N = 4), suggesting that the effect of NO was through cGMP generation. Nuclear translocation of both phosphorylated kinases was seen after 10 minutes of stretch and was largely prevented by 8-bromo-cGMP. Increased DNA binding of activator protein-1 proteins was observed in the nuclei of stretched MCs at 10 minutes by mobility shift assay (N = 4), which was also largely prevented by 8-bromo-cGMP. Stretch increased PCNA expression by MCs, and this was inhibited by 8-bromo-cGMP. Conclusions. Stretch-induced activation of SAPK and p42/44 MAPK in MCs can be inhibited by NO. The effect of NO is mediated by the generation of cGMP. These mechanisms may be responsible, at least in part, for the protective effect of NO in animal models of glomerular injury characterized by glomerular capillary hypertension.

AB - Background. In vivo, intraglomerular hypertension results in resident cell hypertrophy, proliferation and matrix protein production, leading to glomerulosclerosis. Mesangial cells (MCs) exposed to in vitro stretch also proliferate and produce matrix. We have shown activation of Jun N-terminal kinase/stress-activated protein kinase (SAPK) and p42/44 mitogen-activated protein kinase (MAPK) in stretched MCs and have also demonstrated that L-arginine decreases resident cell proliferation and protects against glomerulosclerosis in remnant kidney glomeruli, presumably by increasing nitric oxide (NO) production. Consequently, we studied whether NO could affect SAPK and p42/44 MAPK activation in stretched MCs. Methods. MCs (passages 5 to 10) cultured on type 1 collagen-coated, flexible-bottom plates were exposed to 0 to 30 minutes of cyclic strain (60 cycles per minute) by computer-driven generation of vacuum of -27 kPa, inducing 28% elongation in the diameter of the surface. Control MCs were grown on coated, flexible-bottom plates. Protein levels (by Western blot) and activity assays for SAPK/JNK and p42/44 MAPK were performed under these conditions. As maximal activation was at 10 minutes, with decay by 30 minutes, the effect of NO on kinase activation was studied at 0, 2, 5, and 10 minutes by preincubation with 70 μmol/L s-nitroso-n-acetylpenicillamine (SNAP; an NO donor) or 1 mmol/L 8-bromo cyclic guanosine monophosphate (8-bromo-cGMP). Downstream events in response to stretch and NO were studied at the time of maximal response (10 minutes) by examining nuclear translocation of SAPK with immunofluorescence microscopy and transcription factor activator protein-1 nuclear protein binding by gel mobility shift assay. The effect of kinase inhibition by NO donors on MC proliferation was studied by Western blotting for proliferating cell nuclear antigen (PCNA). Results. Cyclic MC stretch led to prompt SAPK and p42/44 MAPK activation, which was maximal at 10 minutes. Preincubation with either SNAP or 8-bromo-cGMP decreased this by 50 and 70%, respectively (N = 4), suggesting that the effect of NO was through cGMP generation. Nuclear translocation of both phosphorylated kinases was seen after 10 minutes of stretch and was largely prevented by 8-bromo-cGMP. Increased DNA binding of activator protein-1 proteins was observed in the nuclei of stretched MCs at 10 minutes by mobility shift assay (N = 4), which was also largely prevented by 8-bromo-cGMP. Stretch increased PCNA expression by MCs, and this was inhibited by 8-bromo-cGMP. Conclusions. Stretch-induced activation of SAPK and p42/44 MAPK in MCs can be inhibited by NO. The effect of NO is mediated by the generation of cGMP. These mechanisms may be responsible, at least in part, for the protective effect of NO in animal models of glomerular injury characterized by glomerular capillary hypertension.

KW - Cell hypertrophy

KW - Glomerulosclerosis

KW - Jun N-terminal kinase

KW - Mechanical strain

KW - Stress-activated protein kinase

UR - http://www.scopus.com/inward/record.url?scp=0033855434&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033855434&partnerID=8YFLogxK

U2 - 10.1046/j.1523-1755.2000.00264.x

DO - 10.1046/j.1523-1755.2000.00264.x

M3 - Article

C2 - 10972671

AN - SCOPUS:0033855434

VL - 58

SP - 1067

EP - 1077

JO - Kidney International

JF - Kidney International

SN - 0085-2538

IS - 3

ER -