MAPK Usage in Periodontal Disease Progression

In periodontal disease, host recognition of bacterial constituents, including lipopolysaccharide (LPS), induces p38 MAPK activation and subsequent inflammatory cytokine expression, favoring osteoclastogenesis and increased net bone resorption in the local periodontal environment. In this paper, we discuss evidence that the p38/MAPK-activated protein kinase-2 (MK2) signaling axis is needed for periodontal disease progression: an orally administered p38α inhibitor reduced the progression of experimental periodontal bone loss by reducing inflammation and cytokine expression. Subsequently, the significance of p38 signaling was confirmed with RNA interference to attenuate MK2-reduced cytokine expression and LPS-induced alveolar bone loss. MAPK phosphatase-1 (MKP-1), a negative regulator of MAPK activation, was also critical for periodontal disease progression. In MPK-1-deficient mice, p38-sustained activation increased osteoclast formation and bone loss, whereas MKP-1 overexpression dampened p38 signaling and subsequent cytokine expression. Finally, overexpression of the p38/MK2 target RNA-binding tristetraprolin (TTP) decreased mRNA stability of key inflammatory cytokines at the posttranscriptional level, thereby protecting against periodontal inflammation. Collectively, these studies highlight the importance of p38 MAPK signaling in immune cytokine production and periodontal disease progression

MKK3/6-p38 MAPK Signaling Is Required for IL-1β and TNF-α-Induced RANKL Expression in Bone Marrow Stromal Cells

Coupled bone turnover is directed by the expression of receptor-activated NF-κB ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). Proinflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induce RANKL expression in bone marrow stromal cells. Here, we report that IL-1β and TNF-α-induced RANKL requires p38 mitogen-activating protein kinase (MAPK) pathway activation for maximal expression. Real-time PCR was used to assess the p38 contribution toward IL-1β and TNF-α-induced RANKL mRNA expression. Steady-state RANKL RNA levels were increased approximately 17-fold by IL-1β treatment and subsequently reduced ∼70%–90% when p38 MAPK was inhibited with SB203580. RANKL mRNA stability data indicated that p38 MAPK did not alter the rate of mRNA decay in IL-1β-induced cells. Using a RANKL-luciferase cell line receptor containing a 120-kB segment of the 5' flanking region of the RANKL gene, reporter expression was stimulated 4–5-fold by IL-1β or TNF-α treatment. IL-1β-induced RANKL reporter expression was completely blocked with specific p38 inhibitors as well as dominant negative mutant constructs of MAPK kinase-3 and -6. In addition, blocking p38 signaling in bone marrow stromal cells partially inhibited IL-1β and TNF-α-induced osteoclastogenesis in vitro. Results from these studies indicate that p38 MAPK is a major signaling pathway involved in IL-1β and TNF-α-induced RANKL expression in bone marrow stromal cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63223/1/jir.2006.26.719.pd

Differential expression of mitogen activating protein kinases in periodontitis

Aim Following toll‐like receptor ( TLR ) engagement, lipopolysaccharide ( LPS ) can stimulate the expression of pro‐inflammatory cytokines thus activating the innate immune response. The production of inflammatory cytokines results, in part, from the activation of kinase‐induced signalling cascades and transcriptional factors. Of the four distinct classes of mitogen‐activated protein kinases ( MAPK ) described in mammals, p38, c‐Jun N‐terminal activated kinases ( JNK 1‐3) and extracellular activated kinases ( ERK 1,2) are the best studied. Previous data have established that p38 MAPK signalling is required for inflammation and bone loss in periodontal disease pre‐clinical animal models. Materials & Methods In this study, we obtained healthy and diseased periodontal tissues along with clinical parameters and microbiological parameters. Excised fixed tissues were immunostained with total and phospho‐specific antibodies against p38, JNK and ERK kinases. Results Intensity scoring from immunostained tissues was correlated with clinical periodontal parameters. Rank correlations with clinical indices were statistically significantly positive ( p ‐value < 0.05) for total p38 (correlations ranging 0.49–0.68), phospho‐p38 (range 0.44–0.56), and total ERK (range 0.52–0.59) levels, and correlations with JNK levels also supported association (range 0.42–0.59). Phospho‐ JNK and phospho‐ ERK showed no significant positive correlation with clinical parameters of disease. Conclusion These data strongly implicate p38 MAPK as a major MAPK involved in human periodontal inflammation and severity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98997/1/jcpe12123.pd

Interactions between extracellular signal‐regulated kinase 1/2 and P38 Map kinase pathways in the control of RUNX2 phosphorylation and transcriptional activity

RUNX2, a key transcription factor for osteoblast differentiation, is regulated by ERK1/2 and p38 MAP kinase‐mediated phosphorylation. However, the specific contribution of each kinase to RUNX2‐dependent transcription is not known. Here we investigate ERK and p38 regulation of RUNX2 using a unique P‐RUNX2‐specific antibody. Both MAP kinases stimulated RUNX2 Ser319 phosphorylation and transcriptional activity. However, a clear preference for ERK1 versus p38α/β was found when the ability of these MAPKs to phosphorylate and activate RUNX2 was compared. Similarly, ERK1 preferentially bound to a consensus MAPK binding site on RUNX2 that was essential for the activity of either kinase. To assess the relative contribution of ERK1/2 and p38 to osteoblast gene expression, MC3T3‐E1 preosteoblast cells were grown in control or ascorbic acid (AA)‐containing medium ± BMP2/7. AA‐induced gene expression, which requires collagen matrix synthesis, was associated with parallel increases in P‐ERK and RUNX2‐S319‐P in the absence of any changes in P‐p38. This response was blocked by ERK, but not p38, inhibition. Significantly, in the presence of AA, BMP2/7 synergistically stimulated RUNX2 S319 phosphorylation and transcriptional activity without affecting total RUNX2 and this response was totally dependent on ERK/MAPK activity. In contrast, although p38 inhibition partially blocked BMP‐dependent transcription, it did not affect RUNX2 S319 phosphorylation, suggesting the involvement of other phosphorylation sites and/or transcription factors in this response. Based on this work, we conclude that extracellular matrix and BMP regulation of RUNX2 phosphorylation and transcriptional activity in osteoblasts is predominantly mediated by ERK rather than p38 MAPKs. © 2012 American Society for Bone and Mineral Research.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90254/1/561_ftp.pd

Expansion of myeloid-derived suppressor cells contributes to metabolic osteoarthritis through subchondral bone remodeling

Background: Osteoarthritis (OA) subsequent to acute joint injury accounts for a significant proportion of all arthropathies. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells classically known for potent immune-suppressive activity; however, MDSCs can also differentiate into osteoclasts. In addition, this population is known to be expanded during metabolic disease. The objective of this study was to determine the role of MDSCs in the context of OA pathophysiology. Methods: In this study, we examined the differentiation and functional capacity of MDSCs to become osteoclasts in vitro and in vivo using mouse models of OA and in MDSC quantitation in humans with OA pathology relative to obesity status. Results: We observed that MDSCs are expanded in mice and humans during obesity. MDSCs were expanded in peripheral blood of OA subjects relative to body mass index and in mice fed a high-fat diet (HFD) compared to mice fed a low-fat diet (LFD). In mice, monocytic MDSC (M-MDSC) was expanded in diet-induced obesity (DIO) with a further expansion after destabilization of the medial meniscus (DMM) surgery to induce post-traumatic OA (PTOA) (compared to sham-operated controls). M-MDSCs from DIO mice had a greater capacity to form osteoclasts in culture with increased subchondral bone osteoclast number. In humans, we observed an expansion of M-MDSCs in peripheral blood and synovial fluid of obese subjects compared to lean subjects with OA. Conclusion: These data suggest that MDSCs are reprogrammed in metabolic disease, with the potential to contribute towards OA progression and severity

Tristetraprolin Regulates Interleukin-6 Expression Through p38 MAPK-Dependent Affinity Changes with mRNA 3' Untranslated Region

Tristetraprolin (TTP) is a well-characterized, zinc finger-containing, RNA-binding protein. TTP targets tumor necrosis factor alpha for degradation via the 3- untranslated region (3-UTR). Although AU-rich elements (AREs) in the 3-UTR of interleukin-6 (IL-6) mRNA dictate mRNA degradation, the role of TTP in the post-transcriptional regulation of IL-6 gene expression is unclear. Here we used TTP-deficient mice to test the hypothesis that IL-6 expression is influenced by TTP. Genetic and siRNA-mediated knockdown of TTP resulted in increased IL-6 production and overexpression of TTP had the reverse effect. IL-6 and tumor necrosis factor alpha production were elevated after injection of IL-1- in TTP-deficient mice. Further, embryonic fibroblasts from these mice (mouse embryonic fibroblasts) exhibited greater IL-6 mRNA expression and longer half-life than wild-type mouse embryonic fibroblasts. Overexpression of TTP reduced IL-6 3-UTR luciferase reporter activity in an ARE-dependent manner. Proximal and distal regions of the 3-UTR acted synergistically to produce the full repression of TTP. Mutation-based luciferase assays show that ARE2, ARE3, and ARE4 are required for TTP-mediated repression. The constitutively activated p38-MK2 pathway abrogated TTP-mediated repression of IL-6 3-UTR reporter activity. RNA immunoprecipitation assay indicated that the deficiency of p38alpha resulted in the increased affinity of TTP to IL-6 mRNA. Taken together, we propose that TTP downregulates IL-6 gene expression at the post-transcriptional level by targeting ARE elements in the 3-UTR region.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90500/1/jir-2E2010-2E0154.pd

A p38 Mitogen‐Activated Protein Kinase Inhibitor Arrests Active Alveolar Bone Loss in a Rat Periodontitis Model

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141943/1/jper1992.pd

Expression of Drug Targets in Patients Treated with Sorafenib, Carboplatin and Paclitaxel

Introduction: Sorafenib, a multitarget kinase inhibitor, targets members of the mitogen-activated protein kinase (MAPK) pathway and VEGFR kinases. Here we assessed the association between expression of sorafenib targets and biomarkers of taxane sensitivity and response to therapy in pre-treatment tumors from patients enrolled in ECOG 2603, a phase III comparing sorafenib, carboplatin and paclitaxel (SCP) to carboplatin, paclitaxel and placebo (CP). Methods: Using a method of automated quantitative analysis (AQUA) of in situ protein expression, we quantified expression of VEGF-R2, VEGF-R1, VEGF-R3, FGF-R1, PDGF-Rβ, c-Kit, B-Raf, C-Raf, MEK1, ERK1/2, STMN1, MAP2, EB1 and Bcl-2 in pretreatment specimens from 263 patients. Results: An association was found between high FGF-R1 and VEGF-R1 and increased progression-free survival (PFS) and overall survival (OS) in our combined cohort (SCP and CP arms). Expression of FGF-R1 and VEGF-R1 was higher in patients who responded to therapy ((CR+PR) vs. (SD+PD+ un-evaluable)). Conclusions: In light of the absence of treatment effect associated with sorafenib, the association found between FGF-R1 and VEGF-R1 expression and OS, PFS and response might reflect a predictive biomarker signature for carboplatin/paclitaxel-based therapy. Seeing that carboplatin and pacitaxel are now widely used for this disease, corroboration in another cohort might enable us to improve the therapeutic ratio of this regimen. © 2013 Jilaveanu et al

Actinobacillus actinomycetemcomitans Lipopolysaccharide‐Mediated Experimental Bone Loss Model for Aggressive Periodontitis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141900/1/jper0550.pd