Role of <i>α</i>-Tubulin Acetylation and Protein Kinase D2 Ser/Tyr Phosphorylation in Modulation by Ghrelin of <i>Porphyromonas gingivalis</i>-Induced Enhancement in Matrix Metalloproteinase-9 (MMP-9) Secretion by Salivary Gland Cells

Abstract

Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase, and hence its processing between the endoplasmic reticulum (ER), Golgi and trans-Golgi (TGN) network remains under a strict control of factors that affect the microtubule (MT) stabilization, and the recruitment and activation of coat and cargo proteins, including ADP-ribosylation factors (Arfs) and protein kinase D (PKD). Here, we report on the factors implicated in the regulation of MMP-9 secretion by salivary gland acinar cells in response to P. gingivalis LPS, and the effect of hormone, ghrelin. We show that the LPS-elicited induction in MMP-9 secretion is associated with the increase in α-tubulin acetylation and the enhancement in MT stabilization, while the modulatory effect of ghrelin is reflected in a decrease in α-tubulin acetylation. Further, the effect of the LPS occurs in concert with up-regulation in Arf-guanine nucleotide exchange factor (GEF)-mediated Arf1 activation and the TGN recruitment of PKD2, while ghrelin exerts the modulatory effect on Arf-GEF activation. Moreover, we reveal that the LPS-induced up-regulation in MMP-9 secretion is reflected in a marked increase in PKCδ-mediated PKD2 phosphorylation on Ser, while the modulatory effect of ghrelin is manifested by the SFK-PTKs-dependent phosphorylation of PKD2 on Tyr. The findings demonstrate that MT stabilization along with Arf-GEF-mediated Arf1/PKD2 activation play a major role in P. gingivalis LPS-induced up-regulation in salivary gland acinar cell MMP-9 secretion, and point the modulatory mode of action by ghrelin.

Role of Signal-Regulated Changes in Microtubule Stability Dynamics through <i>α</i>-Tubulin Phosphorylation on Ser/Tyr in Modulation of Salivary Gland Matrix Metalloproteinase-9 (MMP-9) Secretion in Response to <i>Porphyromonas gingivalis</i> and Ghrelin

“…Considering the glycoprotein nature of MMP-9 and the fact that protein glycosylation occurs mainly in the Golgi-TGN [7] [31], the above findings lend further support to the earlier data [23], suggesting that P. gingivalis exerts its effect on the acinar cell MMP-9 secretion at the level of ER-to-Golgi trafficking that requires Arf1/PKD2 participation, and that the extent of MT stabilization plays a regulatory role in the process of Arf1 Thus, our findings underscore the role of signal-regulated changes in MT stability dynamic through α-tubulin phosphorylation in controlling the salivary gland acinar cell MMP-9 secretion in response to P. gingivalis LPS as well as the modulatory influence of ghrelin. The diagram depicting the influence of P. gingivalis LPS and ghrelin on MT dynamics and the acinar cell secretion of MMP-9 is depicted in Figure 7.…”

“…cells with GF109203X led to an enhancement in the inhibitory potential of ghrelin, the SFK-PTKs inhibitor, PP2, caused the suppression in ghrelin effect on the LPS-induced changes in MMP-9 secretion and GTP-Arf1 formation. These findings, thus support the involvement of SFK-PTKs and PKC, identified earlier as PKCδ [23], in the modulation of salivary gland acinar cell MMP-9 secretion and Arf1 activation in response to P. gingivalis LPS.…”

“…The supernatants were precleared with GST beads and the beads were incubated with a 100 µg of GST-GGA3-PBD proteinsPAK1 PBD-agarose for 1 h at 4˚C. The beads were washed three times in the lysis buffer, resuspended in Laemmli reducing sample buffer, resolved on 12% SDS-PAGE, and immunoblotted for GTP-bound Arf1 using anti-Arf1 antibody [23].…”

“…The cells were then resuspended in the medium to a concentration of 2 × 10 7 cell/ml, and transferred in 1 ml aliquots to DMEM in culture dishes and incubated under 95% O 2 and 5% CO 2 at 37˚C for up to 16 h in the presence of 0 -100 ng/ml P. gingivalis LPS [23]. P. gingivalis used for LPS preparation was cultured from clinical isolates obtained from ATCC No.…”

“…The diverse aspects of MT dynamics arise from accumulation of a variety of posttranslational modifications on the tubulin subunits, including acetylation, detyrosination, polyglutamylation, polyglycylation and phosphorylation, and are further tuned and refined through binding of MT-associated proteins [19] [20] [21] [22]. Indeed, changes in MT stability dynamics and the increase in α-tubulin acetylation have been directly linked to the increase in MMP-9 secretion elicited by MT stabilizing agents [17], and we have demonstrated previously that MT destabilizing agents that inhibit microtubule polymerization exert the inhibitory effect on the increase in salivary gland acinar cell MMP-9 secretion elicited by P. gingivalis LPS [23]. Other findings point to the role of tubulin phosphorylation in MT stabilization, and suggest the involvement of protein kinase C (PKC) and Src family tyrosine kinase (SFK) in this process [24] [25] [26] [27].…”