Caspase recruitment domain (CARD) protein expression and activity in airway epithelium. (A) Expression of a subset of CARD-containing genes in human primary bronchial epithelial cells, human airway smooth muscle cells, Jurkat, and 293 cells. Cells were transfected with constructs expressing the indicated genes. QPCR was preformed using RNA isolated from the cells. GAPDH was used to normalize the values of CARD genes tested. (B) NF-κB activation mediated by expression of a series of CARD genes in A549 cells, as measured by luciferase activation. All experiments were performed at least two times.

CARMA3 is expressed in murine airway epithelial cells. (A) CARMA1, CARMA2, and CARMA3 RNA levels measured by QPCR and normalized to GAPDH in cell-types isolated and cultured from the lungs of mice. MTE, mouse tracheal epithelial cells; DC, dendritic cell; EC, endothelial cell. (B) Immunohistochemistry of normal mouse lung with stained with an antibody to CARMA3. There is impressive staining of bronchial epithelium.

Thymic stromal lymphopoietin (TSLP) and CCL20 expression in normal human epithelial cells in response to LPA stimulation. (A and B) Normal human bronchial epithelial (NHBE) cells were grown on an air–liquid interface and stimulated with 10 μM lysophosphatidic acid (LPA) for the indicated time period. RNA was isolated from the cells and the levels of (A) TSLP and (B) CCL20 were determined by QPCR. Values are the mean of three to six samples ± SEM. *P < 0.05 versus unstimulated cells by unpaired t test. This experiment was repeated two times. (C and D) NHBE cells grown on an air–liquid interface were stimulated with increasing concentrations of LPA or sphongosine-1-phosphate (S1P) for 6 hours. RNA was isolated from the cells and the levels of (C) TSLP and (D) CCL20 were determined by QPCR. Values are the mean of three to six samples ± SEM. *P < 0.05 versus S1P stimulated cells by ANOVA. This experiment was repeated two times. (E) NHBE cells were grown on an air–liquid interface and stimulated for 6 hours with media alone (serum-free), 10 ng/ml TNF-α, 1 ng/ml IL-1β, or 10 μM LPA. TSLP expression was then analyzed by commercial enzyme-linked immunosorbent assay (ELISA). Values are the mean of four samples ± SEM. *P < 0.05 versus unstimulated cells by unpaired t test.

CARMA3 mediates LPA-induced NF-κB activity and TSLP expression. (A) MTE, NHBE, and A549 cells were grown in culture for 1 to 2 weeks and then harvested for RNA isolation. CARMA3 RNA levels were measured by QPCR and normalized to GAPDH in the indicated cell types. (B) A549 cells were transfected with WT CARMA3, the L51R dominant-negative mutant form of CARMA3, or a control vector plus NF-κB luciferase reporter and Renilla plasmids. NF-κB activity in these cells was assessed by normalization of firefly luciferase activity to Renilla luciferase activity at baseline and after incubation for 6 hours with 10 μM LPA. This experiment was repeated two times. (C) TSLP RNA levels measured by QPCR and normalized to GAPDH in transfected A549 cells at baseline and after incubation for 6 hours with 10 μM LPA. Values are the mean of three samples ± SEM. *P < 0.05 by unpaired t test. This experiment was repeated two times.

Knockdown of CARMA3 attenuates LPA induced TSLP and CCL20 expression in NHBE cells. (A) A549 cells were transfected with lentiviral vectors expressing shRNA sequences specific to CARMA3 or a control vector. CARMA3 RNA knockdown was assessed by measuring the level of CARMA3 expression by QCPR. Levels were normalized to A549 cells transfected with a control vector and expressed as relative expression compared with control levels. Values are the mean of three samples ± SEM. Western blot of cell lysates from A549 cells transfected with WT CARMA3 and constructs containing sequences for shRNA against CARMA3. Blots were probed with an antibody to Myc. (B) CARMA3 RNA levels measured by QPCR and normalized to GAPDH in NHBE cells after transfection with a construct (shRNA48) containing an shRNA specific to CARMA3 or a control vector. Values are the mean of three samples ± SEM. This experiment was repeated two times. (C) TSLP and CCL20 RNA levels measured by QPCR and normalized to GAPDH in transfected NHBE cells at baseline and after incubation for 6 hours with 10 μM LPA. There are at least six samples in each group pooled from two experiments. *P < 0.05 by unpaired t test. (D) TSLP and CCL20 protein levels in culture supernatants measured by ELISA in transfected NHBE cells at baseline and after incubation for 6 hours with 10 μM LPA. There are at least three samples in each group pooled from two experiments. *P < 0.05 by unpaired t test.