CCK Receptor And Anti-inflammatory Intracellular Signaling Mechanisms Activated By CCK-8

The uncontrolled inflammatory response resulted by endotoxin-induced overproduction of inflammatory mediators is considered as the major factor in the pathogenesis of infectious or traumatic diseases complicated with multiple organ dysfunction syndrome (MODS). It is well known that the activation of monocytes/macrophages plays a critical role in inducing the inflammatory response. Macrophages stimulated by LPS (main component of endotoxin) or other inflammatory factors produce and release large quantity of various proinflammatory cytokines including TNF-α, IL-1β, IL-6. Overproduction of the cytokines can result in systemic inflammatory response syndrome (SIRS), MODS and death. The lung is highly sensitive to LPS to be dysfunctional, suggesting that pulmonary macrophages activated by LPS release some mediators to produce the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Pulmonary macrophages consist of three major subpopulations including alveolar macrophages (AMs) located primarily in the alveolar cavity, pulmonary interstitial macrophages (PIMs) which reside within the lung parenchyma, and pulmonary intravascular macrophages. It was reported that there was a significant increase in production of oxygen free radicals from PIMs, but not from AMs in the rats treated with LPS. The treatment also markedly enhanced phagocytosis only in PIMs and caused a significant increase in chemotaxis of PIMs toward C5a. These data demonstrate that PIMs play an important role in the inflammatory response of the lungs in acute endotoxemia.CCK is discovered initially in the gut as a gastrointestinal hormone with the function of contracting gallbladder and mediating pancreatic secretion, and subsequently localized in the central and peripheral nervous system as a neurotransmitter or neuromodulator to modulate dopamine release and reward.CCK is identified as several different size of the peptide including 4, 8, 33, 39, and 58 amino acid forms. The sulfated carboxyl-terminal octapeptide (sCCK-8) is the biologically predominant active form localized in the small intestine, blood and CNS. For recent years, a series of studies focused on the effect of CCK-8 against endotoxin shock and inflammation have being performed in our laboratory. A lot of data demonstrate that sCCK-8 causes an in vitro inhibition of LPS-induced TNF-a production, sCD14 release and mCD14 expression in rat PIMs. Consistently, the production of proinflammatory cytokines including TNF-a, IL-lf3 and IL-6 in endotoxin shock (ES) rat was also inhibited by sCCK-8 in vivo. These results suggested that sCCK-8 has anti-inflammatory effect to some extent, a new field about the biological action of CCK, which was also confirmed by a morphological observation that sCCK-8 clearly lessened the inflammatory lesions in lung, spleen and liver tissues in ES rat. However, the anti-inflammatory mechanism and signal transduction of sCCK-8 remain unclear. It is well known that CCK exerts a variety of physiological actions through its cell surface receptors, which have been pharmacologically classified into two subtypes CCK-AR and CCK-BR according to their affinity to the peptide agonist sCCK-8 and gastrin. CCK receptors belong to G protein-coupled receptor (GPCR) superfamily and distribute extensively in mammalian body. More recently, some studies of our laboratory proved the presence of CCK-AR and CCK-BR mRNA expression in rat spleen cells, lung and heart tissues, and the expression could be up-regulated by LPS obviously. The results indicate that CCK-8 might bind to CCK receptors and interfere with the over activation of immune cells induced by LPS to exert its anti-inflammatory effect during endotoximia.It is well known that a number of signaling pathways are involved in activation of macrophages induced by LPS and there is a lot of cross talk between them. The LPS-induced cytokine production and release require the activation of a set of multiple transcription factors including NF-kB, STAT1, AP-1 and others. However, the most important transcription factor, which is involved in the regulation of nearly all genes expressed in response toinflammation, appears to be NF-kB. NF-kB belongs to NF-KB/Rel family of dimeric transcription factors which are sequestered in the cytosol by the inhibitory protein I-kB. Phosphorylation of I-kB leads to its ubiquityinylation and subsequent degradation resulting in NF-kB activation and translocation into the nucleus to initiate gene expression. PKC-regulated signaling pathways are linked to the upstream activation mechanisms of NF-kB, especially, recent findings showed that PKC^ can regulate NF-kB via an IKK-independent pathway, by directly phosphorylating Ser3" of the p65 subunit (RelA), and play a pivotal role in mediating inflammatory response induced by LPS.To elucidate the anti-inflammatory intracellular signaling mechanism activated by CCK-8, we performed a series of studies on rat PIMs, from the expression of CCK receptor mRNA, the effect of CCK-8 on DAG-PKC pathway and transcription factors involved in regulating inflammatory response induced by LPS to the crosstalk between cAMP-PKA and DAG-PKC signaling pathways.1 Expression of CCK receptor mRNA and effect of LPS on its expression in rat PIMsThe PIMs isolated from rat lung tissues were purified by the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion, and incubated with LPS (lOmg/L) in different time. The expression subtypes of CCK receptors were detected and the effects of LPS on its expression were investigated by RT-PCR and Southern blot. Data were presented as x + s and analyzed by one way ANOVA and least significant difference test using SPSS statistical program. A level of p<0.05 was considered statistically significant. Results: (1) CCK-AR and CCK-BR mRNA were detected in PIMs isolated from rat lung tissues by means of RT-PCR, and their amplification products had a size of approximately 1.37kb and 480bp, respectively. Amplification of f3-actin was always involved to serve as control of reaction efficacy and the products were of 420bp length. (2) The relative expressed quantity (ratio of arbitrary unit over P-actin) of CCK-AR was (31.2 ±3.56)%in control group and (36.7±4.38)%, (59.2±3.41)%, (52.7±2.84)%,(51.3±3.15)%in LPS 0.5h, 2h, 6h and 12h, respectively, and significant increases in its expression were observed at indicate time after incubating rat PIMs with LPS (p<0.0\), but with an exception of which at 0.5h O>0.05). For CCK-BR mRNA, the relative expressed quantity was (34.6±3.24)%in control group and (58.2±3.56)%, (79.6±3.15)%, (60.7±3.15)%, (54.3±3.46) %in LPS 0.5h, 2h, 6h and 12h, respectively, and the expression level of every time point in LPS group was higher than that of control group(p<0.01). The relative expressed quantity of CCK-BR mRNA was increased in comparison with that of CCK-AR mRNA after incubation with LPS for 0.5h, 2h and 6h (P<0.0\). (3) Southern blot analysis of PCR amplified CCK-AR and CCK-BR mRNA products using [y-32P] ATP 5'-end-labelled probes showed specific hybridization bands. These studies for the first time suggested the presence of CCK-AR and CCK-BR mRNA expression in rat PIMs, which provide structural basis for further elucidation of anti-inflammatory signaling mechanisms activated by CCK-8 during endotoximia, and the expression could be upregulated by LPS obviously.2 Effect of CCK-8 on DAG-PKC signaling pathway in rat PIMs 2.1 Effect of CCK-8 on DAG-PKC signaling pathway in resting rat PIMs We have demonstrated in the first part that PIMs expressed CCK-AR and CCK-BR gene, and the expression could be upregulated by LPS. It is well known that protein kinase C, as an important message molecular, participates immune regulation and inflammatory response signal transduction extensively, particularly, PKC^ plays a pivotal role in activation of monocytes/ macrophages induced by LPS. An increasing amount of data supports the concept that the signal transduction cascades mediated by CCK receptors are cell specific. For example, CCK was found to activate cAMP and PKC regulated signaling pathways in pancreatic acinar cells, but to induce a significant decrease in membrane and cytosol PKC activity in murine lymphocytes, neutrophil and peritoneal macrophages and an increase of intracellular cAMP levels. As little is known about the action of CCK-8 on DAG-PKC signaling pathway, the aim of this study was to investigate theeffect of CCK-8 on DAG content, PKC activity and PKC^ translocation in resting rat PIMs and establish a basis for further elucidation of anti-inflammatory molecular mechanisms activated by CCK-8. PIMs were isolated and incubated with CCK-8 at different concentration for indicated time. DAG content and PKC activity were detected by radioenzymatic assay and PKC^ translocation was determined by immunoblotting. Data were presented as x ± s and analyzed by one way ANOVA and least significant difference test using SPSS statistical program. A level of p<0.05 was considered statistically significant. Results: (1) CCK-8 did not affect DAG content obviously at low concentration (10"!2~10"7mol/L)(p>0.05), but led to a decrease of DAG content at high concentration (10"6~10"5mol/L) compared with that of control group (p<0.0\), suggesting that resting PIMs were not sensitive to stimulation of CCK-8. (2) Treating PIMs with CCK-8, no changes of cytosolic and membrane-bound PKC activities were found at low concentration (10~12~10"9mol/L), but an increase of cytosolic PKC activities and a decrease of membrane-bound PKC activities were observed gradually accompany with the increase of CCK-8 concentration and significant differences presented at 10"6 and 10~5 mol/L (p<0.0\). (3) Immunoblotting analyses showed that CCK-8 inhibited PKCt, translocation from cytosol to membrane only at high concentration (10~6mol/L) (p<0.05). These results demonstrated that CCK-8 was a negative modulator of DAG-PKC signaling pathway at low concentration, which had important significance to maintain body homeostasis, and led to an inhibition of DAG-PKC signaling pathway at high concentration. 2.2 Effect of CCK-8 on DAG-PKC signaling pathway in rat PIMsstimulated by LPSIt is well known that a number of signaling pathways are involved in activation of monocytes/macrophages induced by LPS, including MAPK and PKC pathway, and there is a lot of cross talk among them. PKC pathway plays a central role in the complicated signaling networks induced by LPS. The present study was performed to investigate the effect of CCK-8 on DAG-PKCsignaling pathway in rat PIMs stimulated by LPS. PIMs were isolated and stimulated with LPS in the absence or presence of CCK-8 for indicated time. DAG content and PKC activity were detected by radio- enzymatic assay and PKC^ translocation was determined by immunoblotting. Data were presented as x ±s and analyzed by one way ANOVA and least significant difference test using SPSS statistical program. A level of p0.05). (2) LPS was found to decrease cytosolic PKC activity and increase membrane-bound PKC activity obviously (p<0.01), suggesting that LPS could promote PKC translocation and change its distribution, but did not affect its total activity in rat PIMs. Treating PIMs with CCK-8, did not affect LPS-induced PKC translocation at 10"n~10"10 mol/L, decreased it slightly at 10"9~10"8mol/L (/?>0.05), and significantly inhibited it at 1O'7~1O*5 mol/L (p< 0.01), suggesting that CCK-8 inhibited LPS-induced PKC translocation in dose-dependent manner. (3) Immunoblotting analyses showed that LPS promoted PKC^ translocation from cytosol to membrane obviously (p<0.0\) and CCK-8 inhibited LPS-induced PKC^ translocation significantly at 10'8 and 10'6 mol/L O<0.01). These results demonstrated that CCK-8 inhibited LPS-induced DAG content, PKC activity and PKCt; translocation significantly at supraphysiological concentration, which might be one of the upstream mechanisms to modulate NF-kB activity and exert anti-inflammatory effect for CCK-8. 2.3 Effect of CCK receptor antagonists on DAG-PKC signaling pathwayin rat PIMs stimulated by LPSWe have demonstrated in the previous part that PIMs expressed CCK-AR and CCK-BR gene, and the expression could be upregulated by LPS, and that CCK-8 inhibited activation of DAG-PKC signaling pathway induced by LPSin dose-dependent manner. However, it is unclear that the inhibitory effect of CCK-8 on DAG-PKC signaling pathway induced by LPS if mediated through CCK receptor. This study was under- taken to investigate the effects of non-selective CCK receptor antagonist proglumide, selective CCK-AR antagonist CR-1409 and selective CCK-BR antagonist CR-2945 on LPS-induced activation of DAG-PKC signaling pathway in rat PIMs, which indicate the receptor mechanism of anti- inflammation and anti-shock effect acted by CCK-8. PIMs were isolated and pretreated with CCK receptor antagonists for 10 min prior to addition of LPS (lOmg/L) and CCK-8 (10"6mol/L), and then co-incubated for indicated time. DAG content and PKC activity were detected by radioenzymatic assay and PKCC, translocation was determined by immunoblotting. Data were presented as x + s and analyzed by one way ANOVA and least significant difference test using SPSS statistical program. A level ofp<0.05 was considered statistically significant. Results: (1) LPS-induced increase of DAG content was inhibited obviously by CCK-8 in rat PIMs (p<0.01). The effect of CCK-8 was abrogated to different degree by proglumide, CR-1409 and CR-2945 accompany with the increase of their concentration. Proglumide was so potent that had an obvious effect on CCK-8-resulted inhibition of LPS-induced DAG content increase at low concentration and even a fully reversal of it at high concentration, and a similar effect was found to CR-1409, but not to CR-2945, for CR-2945 showing weak effect only at high concentration. The IC50 of proglumide, CR-1409 and CR-2945 were (1.76±0.19) X 10"6mol/L, (6.76±0.52) X 10"5mol/L and (5.45±0.42)X 10"3 mol/L, respectively. (2) The inhibitory effect of CCK-8 on LPS-induced PKC activity translocation in rat PIMs was attenuated by proglumide, CR-1409 and CR-2945 at 10"4mol/L, and their inversio rate (percent of CCK-8) were 58.9 % , 48.1 % and 21.5 % , respectively (p<0.01). (3) The LPS-induced PKCt, translocation from cytosol to membrane was inhibited by CCK-8 significantly O<0.01). The effect of CCK-8 was blocked by proglumide, CR-1409 (p<0.0l) and CR-2945 (p<0.05) to different degree. These results suggested that the inhibitory effect of CCK-8on LPS-activated DAG-PKC signaling pathway in rat PIMs was mediated through CCK receptor, and that both CCK-AR and CCK-BR might be involved in this pathway, but CCK-AR might play a main role in this processe. 3 Involvememt of PKC signaling pathway in CCK-8 regulation of LPS-induced NF-kB activity in rat PIMsNF-kB plays a pivotal role in LPS-induced TNF-a and IL-lf3 gene expressions, and that PKC signaling pathway is linked to upstream regulation mechanism of NF-kB activation. Previous studies in our laboratory showed that CCK-8 inhibited LPS-induced NF-kB binding activity in a dose-dependent manner and we have demonstrated in part 2 that CCK-8 also inhibited LPS-induced activation of DAG-PKC signaling pathway. But it remains unclear that whether CCK-8 regulates LPS-induced NF-kB binding activity via inhibition of DAG-PKC signaling pathway activation. This study was performed to determine if DAG-PKC signaling pathway was involved in regulation of LPS-induced NF-kB binding activity by CCK-8 in rat PIMs and so that further elucidate the anti-inflammatory molecular mechanisms activated by CCK-8. PIMs were isolated and stimulated with LPS in the absence or presence of CCK-8 and/or PMA (phorbol 12-myristate, 13-acetate, PKC activator), and NF-kB binding activity was analyzed by electrophoretic mobility shift assay (EMSA) lh after stimulation. The protein level of IkB-(x, p-lKB-a and actin in the cytoplasma were detected by Western blot 30 min after stimulation. At the same time, the effects of CCK receptor antagonists on LPS-induced IkB-(x phosphorylation were observed. Data were presented as x ± s and analyzed by one way ANOVA and least significant difference test using SPSS statistical program. A level of p<0.05 was considered statistically significant. Results: (1) The NF-kB binding activity was significantly higher in PIMs stimulated with lOmg/L LPS in comparison with unstimulated cells (p<0.0\), of which was too little to be detected, and additional treatment with CCK-8 markedly reduced the binding activity and the inhibition rate reached to 77.3 %(p<0.0\). CCK-8 alone had no effect on the NF-kB binding activity (p>0.05). PMA, a PKC activator, alone or co-incubated with LPS resulted inobvious increase of NF-kB binding activity (p<0.0\) and treatment with CCK-8 led to a reversal of PMA and/or LPS-induced increase of NF-kB binding activity by 42.3% and 52.4%, respectively (pO.Ol). (2) The IkB-ol protein level was markedly decreased and p-lKB-a protein level was obviously increased in PIMs 30 min after incubation with LPS or/and PMA (p<0.01), and CCK-8 at the concentration of 10"7 mol/L increased IkB-cx and decreased p-lKB-a protein level significantly in PIMs stimulated by LPS or/and PMA (pO.Ol). The effect of CCK-8 on LPS-induced IicB-a phosphorylation was blocked by proglumide and CR-1409 (p<0.0\), but not by CR-2945 (p>0.05). CCK-8 alone had no effect on the protein level of IicB-a and p-lKB-a in PIMs (p>0.05). These results revealed that CCK-8 inhibited NF-kB binding activity and decreased IkB-cx degradation through CCK receptors in rat PIMs stimulated by LPS and DAG-PKC signaling pathway was involved in regulation of NF-kB binding activity by CCK-8 at least in part, which might be one of anti-inflammatory mechanisms activated by CCK-8. 4 Cross-talk between PKA and PKC signaling pathway in rat PIMsWe had demonstrated previously that CCK-8 inhibited LPS-induced activation of DAG-PKC signaling pathway through CCK receptor, and then decreased various proinflammatory cytokines production via regulation of NF-kB activity in rat PIMs, which might be one of anti-inflammatory mechanisms activated by CCK-8. But how and why CCK-8 inhibited LPS-induced activation of DAG-PKC signaling pathway remains unclear. Recently, it has been shown that many of the cellular responses mediated by GPCRs do not involve the sole stimulation of conventional second-messenger-generating systems, but instead result from the functional integration of a intricate network of intracellular signaling pathways. The present study was undertaken to investigate the effect of forskolin (FSK, a cAMP activator) and H-89 (a specific inhibitor of PKA) on LPS-induced activation of DAG-PKC signaling pathway and elucidate the crosstalk between PKA and PKC signaling pathways. PIMs were isolated and stimulated with LPS or/and CCK-8 in the absence or presence of FSK or H-89 for indicated time. DAGcontent and PKC activity translocation were detected by radioenzymatic assay. Data were presented as x+s and analyzed by one way ANOVA and least significant difference test using SPSS statistical program. A level of p<0.05 was considered statistically significant. Results: (1) DAG content in FSK and FSK+CCK group decreased, slightly but not significantly in comparison with control group (p>0.05) in resting rat PIMs; LPS at lOmg/L increased DAG content(><0.01) and FSK or/and CCK-8 decreased LPS-induced DAG content obviously(p<0:05, /?<0.01), but H-89, a specific inhibitor of PKA, led to a reversal of CCK-8 resulted inhibition of LPS-induced DAG content(/?<0.01). H-89 alone did not affect LPS-induced DAG content O>0.05). (2) No changes of cytosolic and membrane-bound PKC activities were found when incubating PIMs with FSK, CCK-8+FSK and CCK-8+H-89 (p>0.05). LPS was found to decrease cytosolic PKC activity and increase membrane-bound PKC activity obviously (p<0.0l), suggesting that LPS could promote PKC translocation, but treating PIMs with FSK or/and CCK-8 inhibited LPS-induced PKC translocation obviously (p<0.0\). Similar results were observed that H-89 led to a reversal of CCK-8 resulted inhibition of LPS-induced PKC translocation (p<0.01), but it alone did not affect LPS-induced PKC translocation (p>0.05). These results demonstrated that CCK-8 inhibited LPS-induced activation of DAG-PKC signaling pathway via evoking of cAMP-PKA signaling pathway, indicating the presence of crosstalk between the two signaling pathways.CONCLUSIONSTo elucidate the receptor and anti-inflammatory intracellular signaling mechanism activated by CCK-8, we firstly performed a series of studies, from the expression of CCK receptor mRNA, the effect of CCK-8 on DAG-PKC signaling pathway and transcription factors involved in regulating inflammatory response induced by LPS to the crosstalk between cAMP-PKA and DAG-PKC signaling pathways.1 The present studies for the first time demonstrated the presence of...