CARMA1 acts downstream of PKCθ in mediating PMA-induced synergistic enhancement of TLR-TRAF6-dependent MUC5AC expression.

<p>(<b>A</b>) CARMA1 was expressed in a variety of epithelial cells, such as in human cervix HeLa, colon HM3, airway A549, middle ear HMEEC-1 and primary bronchial epithelial NHBE cells, as assessed by WB using antibody against CARMA1. (<b>B</b>) Overexpression of a DN mutant form of CARMA1 attenuated C/A-PKCθ-induced MUC5AC expression. Cells were transfeced with 0.3 µg of C/A PKCθ with or without 0.8 µg of DN-CARMA1. Relative luciferase activity of MUC5AC was measured from the cell lysate. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. control; <i>**p<0.05</i> vs. C/A PKCθ transfected cells. (<b>C</b>) The synergistic induction of MUC5AC transcription by NTHi and PMA was potently inhibited by overexpression of DN-CARMA1. Cells were transfeced with 0.8 µg of DN-CARMA1 or control vector, and treated with NTHi with or without PMA. Relative luciferase activity of MUC5AC was measured from the cell lysate. Values are the means ± S.D. (n = 3). <i>*p<0.05</i> vs. NTHi alone; <i>**p<0.05</i> vs. control vector transfected cells. (<b>D</b>) Overexpression of a DN mutant form of CARMA1 greatly inhibited not only synergistic phosphorylation of p38 MAPK and MKK3/6 induced by NTHi and PMA, but also ERK and MEK phosphorylation. Cells were transfeced with 0.8 µg of DN-CARMA1 or control vector, and treated with NTHi with or without PMA. Cell lysate was blotted with antibodies indicated in the figure. The data shown are representative of three independent experiments. −, absence of; +, presence of; NTHi, nontypeable <i>Haemophilus influenzae</i>; DN, dominant negative; C/A, constitutively active form.