Focal adhesion kinase-related nonkinase (FRNK) negatively regulates IL-4-mediated inflammation

Abstract

Focal adhesion kinase (FAK)-related nonkinase (PTK2 isoform 6 in humans, hereafter referred to as FRNK) is a cytoskeletal regulatory protein that has recently been shown to dampen lung fibrosis, yet its role in inflammation is unknown. Here, we show for the first time that expression of FRNK negatively regulates IL-4-mediated inflammation in a human model of eosinophil recruitment. Mechanistically, FRNK blocks eosinophil accumulation, firm adhesion and transmigration by preventing transcription and protein expression of VCAM-1 and CCL26. IL-4 activates STAT6 to induce VCAM-1 and CCL26 transcription. We now show that IL-4 also increases GATA6 to induce VCAM-1 expression. FRNK blocks IL-4-induced GATA6 transcription but has little effect on GATA6 protein expression and no effect on STAT6 activation. FRNK can block FAK or Pyk2 signaling and we, thus, downregulated these proteins using siRNA to determine whether signaling from either protein is involved in the regulation of VCAM-1 and CCL26. Knockdown of FAK, Pyk2 or both had no effect on VCAM-1 or CCL26 expression, which suggests that FRNK acts independently of FAK and Pyk2 signaling. Finally, we found that IL-4 induces the late expression of endogenous FRNK. In summary, FRNK represents a novel mechanism to negatively regulate IL-4-mediated inflammation.

Inhibiting focal adhesion kinase (FAK) blocks IL-4 induced VCAM-1 expression and eosinophil recruitment in vitro and in vivo

“…Further studies showed that either FAK siRNA or the dominant negative protein FAK‐related non‐kinase (FRNK) attenuated neutrophil transmigration across TNF‐α stimulated endothelial cells . Recently, we showed that FRNK also blocked eosinophil transmigration; however, the mechanism was different than what we found for neutrophils . In this case, FRNK acted independently of FAK and acted much earlier in the recruitment cascade by preventing the IL‐4‐induced expression of vascular cell adhesion molecule‐1 (VCAM‐1) and CCL26 …”

“…After 24 h, the incubation medium was removed and endothelial cell viability was measured using trypan blue exclusion. The supernatant was assayed for CCL26 using ELISA as previously described . The monolayer was then washed and endothelial cells were harvested for qPCR or Western blotting.…”

“…Exposure of the endothelium to pro‐inflammatory mediators initiates a well‐described series of events that culminates in the expression of adhesion molecules and chemokines that bind and activate leukocytes leading to emigration into the tissue . Over the past decade, endothelial focal adhesion kinase (FAK) has emerged as an important modulator of transmigration …”

“…FAK is a multifunctional molecule known for its roles in vascular development, gene expression, cell migration, junctional integrity, and leukocyte transmigration . During leukocyte recruitment, several groups have shown changes in FAK phosphorylation, localization, and total protein expression . For example, Iwaki et al.…”

mTORC2 Signaling Selectively Regulates the Generation and Function of Tissue-Resident Peritoneal Macrophages

“…It has previously been shown that IL-4 can enhance GATA6 expression at the level of transcription in human umbilical vein endothelial cells (Sharma et al, 2015). Therefore, we hypothesized that mTORC2 signaling was also regulating the expression of GATA6 at the level of mRNA.…”