Narayanan Parameswaran

Adenovirus Vector-Induced Innate Inflammatory Mediators, MAPK Signaling, As Well As Adaptive Immune Responses Are Dependent upon Both TLR2 and TLR9 In Vivo

Adenovirus (Ad) vectors are promising candidates for both gene transfer and vaccine applications. In this study, we investigated the role of TLR2 in innate and adaptive immune responses to Ad and/or the transgene it expresses following systemic injection. We found that Ad directly activates ERK1/2 in vivo, but that initiation of ERK1/2 activation is primarily a MyD88/TLR2-independent, but Kupffer cell-dependent, event. The complexity of Ad-induced innate immune responses was confirmed when we also found that both TLR2 and MyD88 functions are required for the sustained activation of ERK1/2. Although we found that the initial activation of NF-κB by Ads is dependent upon MyD88, but independent of TLR2 in (non-Kupffer cells) the liver, TLR2 significantly influenced the Ad-induced late phase NF-κB activation. These very rapid responses were positively correlated with subsequent innate immune responses to the Ad vector, as our results confirmed that the induction of several cytokines and chemokines, and the expression of innate immune response genes following Ad injection were TLR2 dependent in vivo. The requirement of TLR2 in Ad-induced innate responses also correlated with significantly altered adaptive immune responses. For example, our results demonstrate that the generation of Ad-neutralizing Abs, and anti-transgene-specific Abs elicited subsequent to Ad vector treatments, are both dependent upon TLR2 functionality. Finally, we found that several Ad-induced innate immune responses are dependent on both TLR2 and TLR9. Therefore, this study confirms that several (but not all) Ad-induced innate and adaptive immune responses are TLR dependent.

Probiotic L. reuteri Treatment Prevents Bone Loss in a Menopausal Ovariectomized Mouse Model

Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post‐menopausal bone loss. Recent studies suggest an important role for gut‐bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 (L. reuteri) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri‐treated mice. Consistent with this, L. reuteri suppressed Ovx‐induced increases in bone marrow CD4+ T‐lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identified that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost‐effective approach to reduce post‐menopausal bone loss. J. Cell. Physiol. 229: 1822–1830, 2014.

Arrestin-2 and G Protein-coupled Receptor Kinase 5 Interact with NFκB1 p105 and Negatively Regulate Lipopolysaccharide-stimulated ERK1/2 Activation in Macrophages

Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IκB kinase promotes phosphorylation and degradation of NFκB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IκB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.

G-protein-coupled-receptor kinases mediate TNFα-induced NF-κB signalling via direct interaction with and phosphorylation of IκBα

Tumor necrosis factor-α (TNFα) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the nuclear factor κB (NFκB) signaling pathway particularly in macrophages has been implicated in many diseases. We demonstrate here that G-protein coupled receptor kinase-2 and 5 (GRK2 and 5) regulate TNFα-induced NFκB signaling in Raw264.7 macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibits TNFα-induced IκBα phosphorylation and degradation, NFκB activation, and expression of the NFκB-regulated gene, macrophage inflammatory protein-1β. Consistent with these results, over-expression of GRK2 or 5 enhances TNFα-induced NFκB activity. In addition,we show that GRK2 and 5 interact with IκBα via the N-terminal domain of IκBα and that IκBα isa substrate for GRK2 and 5 in vitro. Furthermore, we also find that GRK5 but not GRK2 phosphorylates IκBα at the same amino acid residues (Ser32/36) as that of IKKβ. Interestingly,associated with these results, knockdown of IKKβ in Raw264.7 macrophages did not affect TNFα-induced IκBα phosphorylation. Taken together, these results demonstrate that both GRK2 and 5 are important and novel mediators of a non-traditional IκBα-NFκB signaling pathway.

Regulation of glomerular mesangial cell proliferation in culture by adrenomedullin.

Adrenomedullin is a recently discovered vasodilatory peptide that has been shown to be a potent activator of adenylate cyclase in a variety of cell systems, including rat mesangial cells. The major aim of the present study was to determine the regulation of rat mesangial cell proliferation (using [3H]thymidine incorporation as an index), apoptosis (using nucleosome-associated cytoplasmic DNA fragmentation as an index) and mitogen-activated protein kinase (MAPK) cascade, specifically extracellular signal-regulated kinase (ERK), jun-amino terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, by adrenomedullin-stimulated cyclic AMP-protein kinase-A pathway. Adrenomedullin increased cAMP levels significantly above basal and the response was inhibited by the adrenomedullin receptor antagonist, adrenomedullin-(22-52). Adrenomedullin also decreased [3H]thymidine incorporation and increased nucleosome-associated cytoplasmic DNA fragmentation, in a concentration-dependent fashion. Both these responses were receptor mediated as, adrenomedullin-(22-52) inhibited these effects. The decrease in proliferation and increase in apoptosis were both mimicked by forskolin, a direct adenylate cyclase activator. Adrenomedullin-mediated decrease in proliferation and increase in apoptosis were inhibited by H89 [[N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride]], a potent protein kinase-A inhibitor. Associated with the changes in proliferation and apoptosis, adrenomedullin decreased ERK2 activity, and increased JNK1 and P38 MAPK activities. All these kinase activities, except the increase in JNK1 activity could be simulated using forskolin. In addition, only adrenomedullin-mediated changes in ERK2 and P38 MAPK activities were inhibited by H89 while, adrenomedullin-stimulated JNK1 was not consistently inhibited by the protein kinase-A inhibitor. These results suggest that adrenomedullin might play an important role in mesangial cell turnover and that although adrenomedullin-mediated responses are primarily cAMP-dependent, it does not preclude the involvement of cAMP-independent pathways.

Receptor Activity-modifying Protein (RAMP) Isoform-specific Regulation of Adrenomedullin Receptor Trafficking by NHERF-1

Receptor activity-modifying proteins (RAMPs 1-3) are single transmembrane accessory proteins critical to various G-protein coupled receptors for plasma membrane expression and receptor phenotype. A functional receptor for the vasodilatory ligand, adrenomedullin (AM), is comprised of RAMP2 or RAMP3 and calcitonin receptor-like receptor (CRLR). It is now known that RAMP3 protein-protein interactions regulate the recycling of the AM2 receptor. The major aim of this study was to identify other interaction partners of RAMP3 and determine their role in CRLR-RAMP3 trafficking. Trafficking of G-protein-coupled receptors has been shown to be regulated by the Na+/H+ exchanger regulatory factor-1 (NHERF-1), an adaptor protein containing two tandem PSD-95/Discs-large/ZO-1 homology (PDZ) domains. In HEK 293T cells expressing the AM2 receptor, the complex undergoes agonist-induced desensitization and internalization. However, in the presence of NHERF-1, although the AM receptor (CRLR/RAMP3) undergoes desensitization, the internalization of the receptor complex is blocked. Overlay assays and mutational analysis indicated that RAMP3 and NHERF-1 interact via a PDZ type I domain on NHERF-1. The internalization of the CRLR-RAMP complex was not affected by NHERF-1 when CRLR was co-expressed with RAMP1 or RAMP2. Mutation of the ezrin/radixin/moesin (ERM) domain on NHERF-1 indicated that NHERF-1 inhibits CRLR/RAMP3 complex internalization by tethering the complex to the actin cytoskeleton. When examined in a primary culture of human proximal tubule cells endogenously expressing the CRLR-RAMP3 complex and NHERF-1, the CRLR-RAMP complex desensitizes but is unable to internalize upon agonist stimulation. Knock-down of either RAMP3 or NHERF-1 by RNA interference technology enabled agonist-induced internalization of the CRLR-RAMP complex. These results, using both endogenous and overexpressed cellular models, indicate a novel function for NHERF-1 and RAMP3 in the internalization of the AM receptor and suggest additional regulatory mechanisms for receptor trafficking.

Toll-like receptors differentially regulate GPCR kinases and arrestins in primary macrophages

G-protein coupled receptor kinases (GRKs) and arrestins (ARRs) are ubiquitously distributed crucial signaling proteins that are critical in the regulation of responsiveness of G-protein coupled receptors (GPCRs). Toll-like receptors (TLRs) (class of pattern recognition receptors) play a vital role in macrophage biology and innate immunity. Because GPCR responsiveness is regulated in part by the expression levels of GRKs/ARRs, the focus of this work was to uncover potential cross-talk mechanisms between TLRs and GPCRs via regulation of GRK/ARR expression in primary mouse macrophages. We demonstrate here that activation of TLR2 and 4 (but not TLR3 and 7) significantly decrease ARR2 but not ARR3 protein levels in macrophages. Compared to this, activation of TLR2, 4, and 7 (but not TLR3) significantly decrease GRK5 and 6 protein levels. Surprisingly, GRK2 protein levels are markedly increased by TLR2, 3, 4 and 7. Mechanistically, expression of ARR2 and GRK5 are regulated at transcriptional as well as post-translational levels. Downregulation of GRK6 by LPS is regulated primarily at the post-translational level. TLR4-induced GRK2 level, however, is both transcriptionally and post-transcriptionally regulated. Our results demonstrate previously unknown crucial regulatory mechanisms that alter ARR/GRK expression levels in macrophages that might modify many, if not all, GPCR-mediated innate immune responses.

Prebiotic and Probiotic Regulation of Bone Health: Role of the Intestine and its Microbiome

Recent advances in our understanding of how the intestinal microbiome contributes to health and disease have generated great interest in developing strategies for modulating the abundance of microbes and/or their activity to improve overall human health and prevent pathologies such as osteoporosis. Bone is an organ that the gut has long been known to regulate through absorption of calcium, the key bone mineral. However, it is clear that modulation of the gut and its microbiome can affect bone density and strength in a variety of animal models (zebrafish, rodents, chicken) and humans. This is demonstrated in studies ablating the microbiome through antibiotic treatment or using germ-free mouse conditions as well as in studies modulating the microbiome activity and composition through prebiotic and/or probiotic treatment. This review will discuss recent developments in this new and exciting area.

Regulation of Lipopolysaccharide-Induced Inflammatory Response and Endotoxemia by β-Arrestins

β‐Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that β‐arrestin‐1 (β‐arr‐1) and ‐2 knockout (KO) mice are protected from TLR4‐mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild‐type (WT) and β‐arr‐1 and ‐2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS‐induced inflammatory cytokine levels in the plasma were markedly decreased in both β‐arr‐1 and ‐2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11b+ and CD11b− populations) from WT, β‐arr‐1, and ‐2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS‐induced inflammatory cytokines were significantly blocked in both splenocyte populations from the β‐arr‐2 KO compared to the WT mice. This effect in the β‐arr‐1 KO mice, however, was restricted to the CD11b− splenocytes. Our studies further indicate that regulation of cytokine production by β‐arrestins is likely independent of MAPK and IκBα‐NFκB pathways. Our results, however, suggest that LPS‐induced chromatin modification is dependent on β‐arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by β‐arrestins in vivo. Taken together, these results indicate that β‐arr‐1 and ‐2 mediate LPS‐induced cytokine secretion in a cell‐type specific manner and that both β‐arrestins have overlapping but non‐redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406–416, 2010.

Adrenomedullin decreases extracellular signal-regulated kinase activity through an increase in protein phosphatase-2A activity in mesangial cells.

Adrenomedullin is a recently identified peptide hormone that has receptors in a number of different systems including renal mesangial cells. We reported recently that adrenomedullin can cause a decrease in extracellular signal-regulated kinase (ERK) activity and increase jun amino-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38 MAPK) acitivities in rat mesangial cells. Associated with these responses we also reported that adrenomedullin can decrease proliferation and increase apoptosis in mesangial cells. The major aim of the present study was to examine the mechanism of decrease in ERK activity by adrenomedullin and to identify the role of protein phosphatase 2A (PP2A) in the decrease in ERK activity, using okadaic acid [9,10-Deepithio-9,10-didehydroacanthifolicin], a selective inhibitor of PP2A at low nanomolar concentrations. The adrenomedullin-induced decrease in [3H]-thymidine incorporation and increase in apoptosis were reversed by okadaic acid at the concentration that selectively inhibits PP2A. Okadaic acid completely reversed the ERK inhibition caused by adrenomedullin, suggesting that PP2A may be involved in the adrenomedullin-mediated changes in proliferation, apoptosis and ERK activity. PP2A activity in mesangial cells was increased over time following exposure to adrenomedullin. The tyrosine phosphorylation of ERK did not change significantly following adrenomedullin treatment although the ERK activity was decreased significantly. This suggests that the decrease in ERK activity is not mediated through a decrease in MEK (a dual phosphorylating kinase upstream of ERK) or by an increase in MKP-1/2 (a dual specificity phosphatase) activities. Thus we conclude that the mechanism of adrenomedullin-induced decrease in ERK activity in rat mesangial cells is at least in part mediated by an increase in PP2A activity.