Effect of protein kinase C and Ca2+ on p42/p44 MAPK, Pyk2, and Src activation in rat conjunctival goblet cells

Abstract

Conjunctival goblet cells synthesize and secrete mucins onto the ocular surface to lubricate it and protect it from bacterial infections. Mucin secretion is under neural control, and cholinergic agonists released from parasympathetic nerves are major stimuli of this secretion. The signal transduction pathways these agonists use to stimulate secretion involve activating protein kinase C (PKC) and increasing intracellular [Ca 2+ ] to activate the non-receptor kinases Pyk2 and p60Src (Src) to transactivate the EGF receptor. Transactivation of the EGF receptor activates a kinase cascade culminating in the activation of p42/p44 MAPK (MAPK) and ultimately that leads to secretion of high molecular weight glycocongujates (HMWGC), including mucins. To further examine the roles of PKC and Ca 2+ in the activation of MAPK, Pyk2, and Src in mucin secretion, rat conjunctival pieces and cultured goblet cells were incubated with the PKC activator phorbol myristate acid (PMA), the cholinergic agonist carbachol, or the calcium ionophore, ionomycin for varying times. Conjunctival pieces were preincubated with PKC inhibitors 10 mins prior to addition of carbachol (10 −4 M) for 10 min. The amount of phosphorylated (activated) MAPK, Pyk2 and Src was determined by western blotting techniques using antibodies specific to the phosphorylated forms of each kinase. PMA significantly increased the activation of MAPK, Pyk2, and Src in a time and concentrationdependent manner. PMA-stimulated MAPK activity was completely inhibited by the EGF receptor inhibitor AG1478 (10 −7 M). Carbachol-stimulated MAPK activity was inhibited by three PKC inhibitors, calphostin C, chelethyrine, and staurosporine. Ionomycin (10 −6 M)-stimulated MAPK activity was inhibited 66% by AG1478 (10 −7 M). Ionomycin also significantly increased Pyk2 and Src in time dependent manner. PKC and ionomycin also activated p42/p44 MAPL, Pyk2, and Src in cultured conjunctival goblet cells. We conclude that PKC and intracellular Ca 2+ activate Pyk2 and Src and phosphorylated the EGF receptor leading to stimulation of MAPK in conjunctival goblet cells. Keywords goblet cells; signal transduction; MAPK; mucin secretionGoblet cells of the conjunctiva are responsible for synthesis, storage, and secretion of mucins, which make up the mucous layer of the tear film (Dartt, 2004, Gipson andArgueso, 2003). Mucins serve to lubricate the ocular surface, protect from bacterial infections and provide for a smooth refractive surface. These cells are highly specialized epithelial cells that are * author to whom correspondence should be addressed: 20 Staniford Street, Boston, MA 02114. Tel: 617-912-7424; FAX: 617-912-0104; email: robin.hodges@schepens.harvard.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form...

A novel Bruton’s tyrosine kinase inhibitor CC-292 in combination with the proteasome inhibitor carfilzomib impacts the bone microenvironment in a multiple myeloma model with resultant antimyeloma activity

“…To confirm that CC-292 does not inhibit c-Src or Pyk2 directly, we next used ionomycin that is known to raise the intracellular level of calcium (Ca 2 þ ), which in turn stimulate c-Src and Pyk2 activity. 39,40 We observed that ionomycin rescued F-actin CC-292 inhibited the OC F-actin ring/sealing zone formation on CaP-coated plates but not on plastic. High concentrations of PF 431396 failed to inhibit F-actin ring/sealing zone formation on plastic, suggesting that the inhibition of F-actin ring/sealing zone formation is observed only on bone surface-like conditions, including dentin or CaP-coated plates.…”

Tear film mucins: Front line defenders of the ocular surface; comparison with airway and gastrointestinal tract mucins

“…Cholinergic agonists activate phospholipase C to increase intracellular [Ca 2+ ] and activate PKC isoforms to cause secretion (Figure 8A) (Dartt et al, 2000; Hodges et al, 2012). The increase in intracellular [Ca 2+ ] and activation of PKC induce Src, PYK2, and ERK1/2 to transactivate the EGFR (Hodges et al, 2007; Horikawa et al, 2003; Kanno et al, 2003). In addition cholinergic agonists activate matrix metalloproteinases of the ADAM family that release EGF or an EGF family member that in turn activates the EGFR.…”

Proteinase-activated receptor 2-mediated calcium signaling in hepatocellular carcinoma cells

“…] i increase results in a complex signaling network that branches into distinct pathways including p42/p44 MAPKinases (Hodges et al 2007;Kanno et al 2003). PAR 2 has been shown recently to mediate p42/p44 MAPKinase activation that has a regulatory function in hepatocellular carcinoma cell invasion (Kaufmann et al 2009).…”

“…Since p42/p44 MAPKinases are known to be regulated by different calcium-dependent mechanisms (Hodges et al 2007;Kanno et al 2003), we investigated the possible contribution of [Ca 2? ] i mobilization in PAR 2 -mediated effect on p42/p44-MAPKinase activation in HCC cells. As demonstrated in Fig.…”