Effect of p38 inhibitors and kinase mutants of the MKK6/p38 signaling module on TNF-–induced MCP-1 promoter activity in HUVEC. (A) Schematic representation of the human MCP-1 promoter/enhancer construct with the SP1 and NF-κB (A1 and A2) sites marked. The proximal promoter and the distal enhancer region are indicated by closed and open boxes. (B) An MCP-1 promoter/enhancer luciferase construct was transfected into HUVEC according to a DEAE-dextran protocol as described in Materials and Methods. After 36 hours, cells were stimulated with 1 ng/mL TNF- for 12 hours with or without a 30-minute pretreatment with 20 μmol/L SB203580 as indicated. (C and D) HUVEC were cotransfected with MCP-1 promoter/enhancer luciferase constructs and kinase mutants MKK6(Ala), MKK6(Glu), p38(TY>AF), or p38(K>M) as indicated. Thirty-six hours later, cells were stimulated with 2 ng/mL TNF-. Relative luciferase activity is expressed as fold stimulation as described in Materials and Methods.