Fractalkine/CX3CL1 production by human airway smooth muscle cells: induction by IFN-gamma and TNF-alpha and regulation by TGF-beta and corticosteroids

Chemokine synthesis by airway smooth muscle cells (ASMC) may be an important process underlying inflammatory cell recruitment in airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Fractalkine (FKN) is a recently described CX(3)C chemokine that has dual functions, serving as both a cell adhesion molecule and a chemoattractant for monocytes and T cells, expressing its unique receptor, CX(3)CR1. We investigated FKN expression by human ASMC in response to the proinflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma, the T helper 2-type cytokines IL-4, IL-10, and IL-13, and the fibrogenic cytokine transforming growth factor (TGF)-beta. Neither of these cytokines alone had any significant effect on ASMC FKN production. Combined stimulation with IFN-gamma and TNF-alpha induced FKN mRNA and protein expression in a time- and concentration-dependent manner. TGF-beta had a significant inhibitory effect on cytokine-induced FKN mRNA and protein expression. Dexamethasone (10(-8)-10(-6) M) significantly upregulated cytokine-induced FKN mRNA and protein expression. Finally, we used selective inhibitors of the mitogen-activated protein kinases c-Jun NH(2)-terminal kinase (JNK) (SP-610025), p38 (SB-203580), and extracellular signal-regulated kinase (PD-98095) to investigate their role in FKN production. SP-610025 (25 microM) and SB-203580 (20 microM), but not PD-98095, significantly attenuated cytokine-induced FKN protein synthesis. IFN-gamma- and TNF-alpha-induced JNK phosphorylation remained unaltered in the presence of TGF-beta but was inhibited by dexamethasone, indicating that JNK is not involved in TGF-beta- or dexamethasone-mediated regulation of FKN production. In summary, FKN production by human ASMC in vitro is regulated by inflammatory and anti-inflammatory factors.     

Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation

Acute hormonalmodulation of NHE3 activity is partly mediated by kinases, includingprotein kinase C (PKC). We examined the role of NHE3 phosphorylation inregulating its activity in response to PKC activation by phorbol12-myristate 13-acetate (PMA). In pooled NHE-deficient fibroblaststransfected with NHE3, PMA increased NHE3 activity and phosphorylation.When six potential PKC target serines were mutated, NHE3phosphorylation was drastically reduced and PMA failed to regulate NHE3phosphorylation or function. To examine whether NHE3 phosphorylation issufficient for functional regulation by PKC, we exploited theheterogeneous response of NHE3 activity to PMA in individual clones oftransfectants. Clones with stimulatory, inhibitory, or null responsesto PMA were observed. Despite the diverse functional response, changesin NHE3 phosphorylation as revealed by tryptic phosphopeptide maps weresimilar in all clones. We conclude that although phosphorylationappears to be necessary, it is insufficient to mediate PKC regulationof NHE3 function and factors extrinsic to the NHE3 protein must be involved.