Figure 8

Mitogen-activated protein kinase (MAPK) family mRNA expression profile in human dermal fibroblasts (a) and human synovial fibroblasts (b) exposed to Staphylococcus aureus whole cell lysate and filtered culture supernatant. Confluent monolayers of de-identified human dermal fibroblasts from normal volunteer and synovial fibroblasts from a patient with rheumatoid arthritis were treated with 25 μg/ml per well of S. aureus whole lysate, filtered culture supernatant, and a combination of rhIL-1β and TNF-α (10 ng/ml each) for 8 hours. Cells were harvested and total cellular RNA was isolated and reverse-transcribed. The MAPK family gene expression was analyzed using the Human MAPK Gene Family I Multigene-12 reverse transcription-polymerase chain reaction (PCR) profiling kit. The semiquantitative values were generated by determining the ratios of the band intensities of respective PCR products to that of housekeeping gene GAPDH (glyceraldehyde phosphate dehydrogenase) in each sample. The experiments were repeated three times. The band densities were determined using three-dimensional densitometric scanning software from Alpha Innotech Corporation (San Leandro, CA, USA). Of the 11 tested Human MAPK Family I genes (ERK1, ERK2, MAPK2/4, ERK3, ERK5, JNK1, JNK2, JNK3, p38b MAPK, p38g MAPK, and p38delta), ERK1, ERK2, JNK1, JNK2, MAPK4, and p38b were elevated significantly in both dermal and synovial fibroblasts upon exposure to S. aureus components (p < 0.05 for individual MAPK gene family member when values from untreated fibroblasts and treated fibroblasts were compared). The response was similar in both cell lines in response to IL-1/TNF. ERK, extracellular signal regulated kinase; IL, interleukin; JNK, c-jun N-terminal kinase; SUP, filtered culture supernatant; TNF, tumor necrosis factor.