Year: 1999
Type: article
Abstract: Hyperglycemia can cause vascular dysfunctions by multiple factors including hyperosmolarity, oxidant formation, and protein kinase C (PKC) activation.We have characterized the effect of hyperglycemia on p38 mitogen-activated protein (p38) kinase activation, which can be induced by oxidants, hyperosmolarity, and proinflammatory cytokines, leading to apoptosis, cell growth, and gene regulation.Glucose at 16.5 mM increased p38 kinase activity in a time-dependent manner compared with 5.5 mM in rat aortic smooth muscle cells (SMC).Mannitol activated p38 kinase only at or greater than 22 mM.High glucose levels and a PKC agonist activated p38 kinase, and a PKC inhibitor, GF109203X, prevented its activation.However, p38 kinase activation by mannitol or tumor necrosis factor-α was not inhibited by GF109203X.Changes in PKC isoform distribution after exposure to 16.5 mM glucose in SMC suggested that both PKC-β2 and PKC-δ isoforms were increased.Activities of p38 kinase in PKC-δ-but not PKC-β1-overexpressed SMC were increased compared with control cells.Activation of p38 kinase was also observed and characterized in various vascular cells in culture and aorta from diabetic rats.Thus, moderate hyperglycemia can activate p38 kinase by a PKC-δ isoform-dependent pathway, but glucose at extremely elevated levels can also activate p38 kinase by hyperosmolarity via a PKC-independent pathway.J. Clin.Invest.103:185-195 (1999). MethodsMaterials.DMEM, FBS, calf serum (CS), transferrin, selenium, Lipofectin and Lipofectamine, and anti-PKC-ε, -γ, -θ, and -ζ antibodies were purchased from GIBCO BRL (Grand Island, New York, USA).Antiphosphospecific p38 MAP kinase antibody and antiphosphospecific MAP kinase kinase (MKK)-3/MKK-6 were obtained from New England Biolabs Inc. (Beverly, Massachusetts, USA).Anti-p38 MAP kinase, ERK-2, PKC-α, JNK, βI, βII, and δ antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA).Antiphosphospecific JNK antibodies and anti-ERK-1 antibodies were obtained from Upstate Biotechnology Inc. (Lake Placid, New York, USA), and [γ-32 P]ATP and [ 3 H]arachidonic acid from Du Pont Nen Research Products (Boston, Massachusetts, USA).The following items were purchased: polyvinylidene difluoride (PVDF) membrane from Novex (San Diego, California, USA); ECL kit from Amersham Life Sciences Inc. (Arlington Heights, Illinois, USA); PMA and bisindolylmaleimide I (GF109203X) from Calbiochem-Novabiochem Corp. (La Jolla, California, USA); recombinant human TNF-α from Pepro Tech Inc. (Rocky Hill, New Jersey, USA); protein A-Sepharose 6MB from Pharmacia Biotech AB (Uppsala, Sweden); protein assay kit from Bio-Rad Laboratories Inc. (Hercules, California, USA); phosphocellulose squares (P-81) from Whatman Institute (Maidstone, United Kingdom); and plasmid maxi kit from QIAGEN Inc. (Valencia, California, USA).LY333351 and cDNA plasmid to PKC-δ isoform was kindly provided by Lilly Research Laboratories (Indianapolis, Indiana, USA), and SB-203580 by SmithKline Beecham Pharmaceuticals (Pittsburgh, Pennsylvania, USA).All other materials were from Fisher Scientific Co. (Pittsburgh, Pennsylvania, USA) and Sigma Chemical Co.(St.Louis, Missouri, USA).Cell culture.Rat aortic smooth muscle cells (SMC) were harvested from male Sprague-Dawley rats (100-150 g) by the media explant technique and cultured in DMEM containing 10% CS.Rat renal mesangial cells were isolated as described previously (16) and cultured in DMEM containing 5 mg/ml each of insulin, transferrin, and selenium with 20% FBS.Human aortic SMC were derived from minced pieces of human aorta and cultured in DMEM containing 20% FBS.Bovine aortic SMC were harvested from calf aorta and cultured in DMEM containing 10% CS.Bovine retinal pericytes were isolated as described previously (41) and cultured in DMEM containing 20% FBS.The cells were used within 10 passages for the following studies. Assay of MAP kinase activity.Subconfluent cultured cells were grown in DMEM containing 10% CS, and then the medium was changed to DMEM containing 1% CS with the indicated concentration of glucose or mannitol.After 48 h of incubation, the cells were incubated in DMEM containing 0.2% BSA with the same concentration of glucose or mannitol for another 24 h.The cells were washed three times with ice-cold PBS containing 1 mM sodium orthovanadate, and then solubilized with 0.5 ml of lysis buffer consisting of 20 mM HEPES, 5 mM MgCl 2 , 25 mM KCl, 1 mM Na 3 SO 4 , 1 mM sodium molybdate, 10 mM β-glycerophosphate, 5 mM tetrasodium pyrophosphate, 250 mM sucrose, 1 mM ATP, 2 mM PMSF, 0.1 mg/ml aprotinin, and 1% Triton X-100.The lysate was centrifuged at 14,000 g to remove insoluble materials, and the supernatant was incubated with anti-ERK-1, anti-ERK-2, or anti-p38 MAP kinase antibody overnight at 4°C.Protein A-Sepharose 6MB was used to precipitate the immune complexes and was incubated for 2 h at 4°C.The pellets were washed twice with ice-cold PBS and the assay buffer consisting of 20 mM MOPS, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM Na 2 SO 4 , and 1 mM DL-dithiothreitol (DTT).Then, 10 µl each of the assay dilution buffer, 2 mg/ml myelin basic protein (MBP), 2 µM protein kinase inhibitor, and a mixture of magnesium/ATP (75 mM MgCl 2 and 500 µM ATP) containing 10 µCi of [γ-32 P]ATP were added to the reaction tube.After 10 min of incubation at room 186
DOI: 10.1172/jci3326
Institutions: Joslin Diabetes Center, Harvard University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2139881033