Table 1.
Primers for RT-PCR analysis.
Fig 1.
CXCL1 and CXCL8 expression in the mucosa specimens.
(A) Immunohistochemical staining of CXCL1 and CXCL8 distribution in the control and CRSsNP nasal mucosa. The inferior turbinates (control) and CRSsNP nasal mucosa from 5 individual patients (pt) were stained with anti-CXCL1 and CXCL8 Ab. Immunoreactivity for CXCL1 and CXCL8 (deep-red staining) was stronger in the submucosal stroma of CRSsNP specimens than in the nasal septal deviation controls. ep: epithelial cells; v: blood vessels; g: submucosal glands. Scale bar = 100 μm.
Fig 2.
Effect of U46619 and IBOP on CXCL1 and CXCL8 expression.
(A) The NMDFs were treated with (a) the indicated concentrations of U46619 for 16 h or (b) U46619 (2 μM) for the indicated time intervals. CXCL1 and CXCL8 in the culture media were analyzed through ELISA. (B) (a) NMDFs were treated with the indicated concentrations of U46619 for 4 h. At the end of the incubation, total RNA was extracted, and the expression of CXC chemokines and GAPDH mRNA was analyzed through RT-PCR and real-time PCR (n = 3).*P < 0.05, **P < 0.01 versus basal (without stimulation). (b) The NMDFs were transfected with CXCL1 promoter luc reporter plasmid for 48 h and followed by U46619 stimulation. The CXCL1 luc reporter activity was determined as described in the Materials and Methods (n = 4). (C) NMDFs were treated with IBOP for (a) 16 h and (b) 8 h. CXCL1 and CXCL8 (a) protein expression was determined through ELISA and (b) mRNA level was determined by RT-PCR and real-time PCR (n = 3).
Fig 3.
TP receptor expression in the CRSsNP mucosa and NMDFs.
(A) Immunohistochemical staining of TP receptor distribution in the control and CRSsNP nasal mucosa. The inferior turbinates (control) and CRSsNP nasal mucosa from 5 individual patients (pt) were stained with the anti-TP receptor Ab. The immunoreactivity for TP receptor was predominantly found in the submucosal stroma of control and CRSsNP specimens. ep: epithelial cells; v: blood vessels; g: submucosal glands. Scale bar = 100 μm (B) TP receptor expression in the NMDFs. Total RNA was extracted from 3 NMDF cultures, and the expression of TPα, TPβ, and β-actin was analyzed through RT-PCR (n = 3).
Fig 4.
Effects of signaling inhibitors on U46619-induced CXCL1 and CXCL8 release and expression.
NMDFs were pretreated with the indicated inhibitors for 30 min and followed by DMSO (-) or U46619 (2 μM) for (A and C) 16 h or (B) 4 h. CXCL1 and CXCL8 release in the culture media was analyzed through ELISA, and their mRNA expression was determined through RT-PCR and real-time PCR. The data are presented as mean ± SEM (n = 3–5). *P < 0.05, **P < 0.01, ***P < 0.001 versus control. LY: 10 μM; Gen: 10 μg/mL; Act D: 2 μM; Tan: 10 μM; PD: 10 μM; GW: 10 μM; H-89: 5 μM; Bay: 5 μM; GF: 2 μM; SQ: 10 μM.
Fig 5.
U46619 induces ERK, PI-3K, PKC, PKA/C substrates, and CREB activation.
NMDFs were treated with U46619 (2 μM) for the indicated time intervals. After incubation, the cell lysates were analyzed through Western blotting (n = 5–7).
Fig 6.
Relationship of PI-3K, PKC, and CREB activation in U44619-induced signaling.
NMDFs were pretreated with SQ29458 (SQ, 10μM), LY294002 (LY, 10 μM), Go6983 (Go, 2 μM), and H-89 (5 μM) for 30 min and followed by stimulation with U46619 (2 μM) for 15 min. (A) A representative blot. (B) Similar results were obtained through densitometry. Data are expressed as mean ± SEM (n = 5–8). *P < 0.05, **P < 0.01 versus U46619 alone.
Fig 7.
Effect of cAMP/PKA-related compounds on CXCL1/8 induction and U46619 on intracellular cAMP production and CREB luc reporter activity.
(A) NMDFs were treated with the indicated compounds for 16 h, and CXCL1 and CXCL8 in the culture medium were analyzed through ELISA. (B) NMDFs were treated with U46619 for 10 min, and intracellular cAMP was assayed through ELISA (n = 4). (C) NMDFs were transfected with the CREB promoter luc reporter for 48 h and followed by U46619 stimulation. CREB luc reporter activity was determined as described in the Materials and Methods (n = 4).*P < 0.05, **P < 0.01 and ***P < 0.001 versus control.
Fig 8.
Effect of CREB, PKCδ, and PKCμ/PKD KD on U46619-induced signaling and CXCL1/8 expression.
(A) Effect of PKCδ/θ and PKCμ/PKD KD on U46619-induced signaling: Cells were transfected with control or PKCδ and PKCμ/PKD siRNA for 72 h and followed by addition of vehicle or U46619 (2 μM) for 120 min. Protein expression and phosphorylation was determined through Western blotting. Two representative results were shown from four independent results. (B) Effect of PKCμ/PKD or CREB KD on U46619-induced CXCL1/8 expression: Cells were transfected with control, PKCμ/PKD, or CREB siRNA for 72 h and followed by addition of vehicle or U46619 (2 μM) for 16 h. CXCL1 and CXCL8 expression was determined through ELISA (n = 3–4). *P < 0.05, **P < 0.01 versus control.
Fig 9.
Proposed mechanism of TXA2-induced CXC chemokine expression in NMDFs.