Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid arthritis (RA), compared to osteoarthritis or other arthritis patients. Several studies suggested an important role for MCP-1 in the massive inflammation at the damaged joint, in part due to its chemotactic and angiogenic effects. It is a known fact that the post-translational modifications (PTMs) of proteins have a significant impact on their properties. In mammals, arginine residues within proteins can be converted into citrulline by peptidylarginine deiminase (PAD) enzymes. Anti-citrullinated protein antibodies (ACPA), recognizing these PTMs, have become a hallmark for rheumatoid arthritis (RA) and other autoimmune diseases and are important in diagnostics and prognosis. In previous studies, we found that citrullination converts the neutrophil attracting chemokine neutrophil-activating peptide 78 (ENA-78) into a potent macrophage chemoattractant. Here we report that both commercially available and recombinant bacterially produced MCP-1/CCL2 are rapidly (partially) degraded upon in vitro citrullination. However, properly glycosylated MCP-1/CCL2 produced by mammalian cells is protected against degradation during efficient citrullination. Site-directed mutagenesis of the potential glycosylation site at the asparagine-14 residue within human MCP-1 revealed lower expression levels in mammalian expression systems. The glycosylation-mediated recombinant chemokine stabilization allows the production of citrullinated MCP-1/CCL2, which can be effectively used to calibrate crucial assays, such as modified ELISAs.

Interleukin 6/Wnt interactions in rheumatoid arthritis: interleukin 6 inhibits Wnt signaling in synovial fibroblasts and osteoblasts.

AIM: To evaluate the impact of previously unrecognized negative interaction between the Wnt and interleukin (IL) 6 signaling pathways in skeletal tissues as a possible major mechanism leading to age- and inflammation-related destruction of bone and joints. METHODS: Luciferase reporter assays were performed to monitor Wnt pathway activation upon IL-6 and tumor necrosis factor-α (TNFα) treatment. Functional contribution of IL-6 and TNFα interaction to inhibition of bone formation was evaluated in vitro using small hairpin RNAs (shRNA) in mouse mesenchymal precursor cells (MPC) of C2C12 and KS483 lines induced to differentiate into osteoblasts by bone morphogenetic proteins (BMP). RESULTS: IL-6 inhibited the activation of Wnt signaling in primary human synoviocytes, and, together with TNFα and Dickkopf-1, inhibited the activation of Wnt response. ShRNA-mediated knockdown of IL-6 mRNA significantly increased early BMP2/7-induced osteogenesis and rescued it from the negative effect of TNFα in C2C12 cells, as well as intensified bone matrix mineralization in KS483 cells. CONCLUSION: IL-6 is an important mediator in the inhibition of osteoblast differentiation by TNFα, and knockdown of IL-6 partially rescues osteogenesis from the negative control of inflammation. The anti-osteoblastic effects of IL-6 are most likely mediated by its negative interaction with Wnt signaling pathway.

Nuclear cytokine-activated IKKalpha controls prostate cancer metastasis by repressing Maspin.

Inflammation enhances tumour promotion through NF-kappaB-dependent mechanisms. NF-kappaB was also proposed to promote metastatogenesis through epithelial-mesenchymal transition. Yet a mechanistic link between inflammation and metastasis is missing. We identified a role for IkappaB kinase alpha (IKKalpha), activated by receptor activator of NF-kappaB (RANK/TNFRSF11A), in mammary epithelial proliferation during pregnancy. Owing to similarities between mammary and prostate epithelia, we examined IKKalpha involvement in prostate cancer and its progression. Here we show that a mutation that prevents IKKalpha activation slows down CaP growth and inhibits metastatogenesis in TRAMP mice, which express SV40 T antigen in the prostate epithelium. Decreased metastasis correlated with elevated expression of the metastasis suppressor Maspin, the ablation of which restored metastatic activity. IKKalpha activation by RANK ligand (RANKL/TNFSF11) inhibits Maspin expression in prostate epithelial cells, whereas repression of Maspin transcription requires nuclear translocation of active IKKalpha. The amount of active nuclear IKKalpha in mouse and human prostate cancer correlates with metastatic progression, reduced Maspin expression and infiltration of prostate tumours with RANKL-expressing inflammatory cells. We propose that tumour-infiltrating RANKL-expressing cells lead to nuclear IKKalpha activation and inhibition of Maspin transcription, thereby promoting the metastatic phenotype.

Histone deacetylase inhibitors suppress rheumatoid arthritis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay.

BACKGROUND: Histone deacetylase inhibitors (HDACi) display potent therapeutic efficacy in animal models of arthritis and suppress inflammatory cytokine production in rheumatoid arthritis (RA) synovial macrophages and tissue. OBJECTIVES: To determine the molecular mechanisms contributing to the suppressive effects of HDACi on RA synovial cell activation, using interleukin 6 (IL-6) regulation as a model. METHODS: RA fibroblast-like synoviocytes (FLS) and healthy donor macrophages were treated with IL-1ß, tumour necrosis factor (TNF)α, lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C)) in the absence or presence of the HDACi trichostatin A (TSA) or ITF2357 (givinostat). IL-6 production and mRNA expression was measured by ELISA and quantitative PCR (qPCR), respectively. Protein acetylation and the activation of intracellular signalling pathways were assessed by immunoblotting. The DNA-binding activity of nuclear factor κB (NFκB) and activator protein 1 (AP-1) components was measured by ELISA-based assays. RESULTS: HDACi (0.25-1.0 µM) suppressed RA FLS IL-6 production induced by IL-1ß, TNFα and Toll-like receptor ligands. Phosphorylation of mitogen-activated protein kinases and inhibitor of κBα (IκBα) following IL-1ß stimulation were unaffected by HDACi, as were AP-1 composition and binding activity, and c-Jun induction. TSA induced a significant reduction in nuclear retention of NFκB in FLS 24 h after IL-1ß stimulation, but this did not reduce NFκB transcriptional activity or correlate temporally with reductions in IL-6 mRNA accumulation. HDACi significantly reduced the stability of IL-6 mRNA in FLS and macrophages. CONCLUSIONS: Our study identifies a novel, shared molecular mechanism by which HDACi can disrupt inflammatory cytokine production in RA synovial cells, namely the promotion of mRNA decay, and suggests that targeting HDAC activity may be clinically useful in suppressing inflammation in RA.

LncCDH5-3:3 Regulates Apoptosis, Proliferation, and Aggressiveness in Human Lung Cancer Cells.

(1) Lung cancer (both small cell and non-small cell) is the leading cause of new deaths associated with cancers globally in men and women. Long noncoding RNAs (lncRNAs) are associated with tumorigenesis in different types of tumors, including lung cancer. Herein, we discuss: (1) An examination of the expression profile of lncCDH5-3:3 in non-small cell lung cancer (NSCLC), and an evaluation of its functional role in lung cancer development and progression using in vitro models; (2) A quantitative real-time polymerase chain reaction assay that confirms lncCDH5-3:3 expression in tumor samples resected from 20 NSCLC patients, and that shows its statistically higher expression levels at stage III NSCLC, compared to stages I and II. Moreover, knockout (KO) and overexpression, as well as molecular and biochemical techniques, were used to investigate the biological functions of lncCDH5-3:3 in NSCLC cells, with a focus on the cells' proliferation and migration; (3) The finding that lncCDH5-3:3 silencing promotes apoptosis and probably regulates the cell cycle and E-cadherin expression in adenocarcinoma cell lines. In comparison, lncCDH5-3:3 overexpression increases the expression levels of proliferation and epithelial-to-mesenchymal transition markers, such as EpCAM, Akt, and ERK1/2; however, at the same time, it also stimulates the expression of E-cadherin, which conversely inhibits the mobility capabilities of lung cancer cells; (4) The results of this study, which provide important insights into the role of lncRNAs in lung cancer. Our study shows that lncCDH5-3:3 affects important features of lung cancer cells, such as their viability and motility. The results support the idea that lncCDH5-3:3 is probably involved in the oncogenesis of NSCLC through the regulation of apoptosis and tumor cell metastasis formation.

Distinct modes of inhibition by sclerostin on bone morphogenetic protein and Wnt signaling pathways.

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.

IKKalpha is a critical coregulator of a Smad4-independent TGFbeta-Smad2/3 signaling pathway that controls keratinocyte differentiation.

Cell-cycle exit and differentiation of suprabasal epidermal keratinocytes require nuclear IkappaB kinase alpha (IKKalpha), but not its protein kinase activity. IKKalpha also is a suppressor of squamous cell carcinoma (SCC), but its mode of action remains elusive. Postulating that IKKalpha may serve as a transcriptional regulator in keratinocytes, we searched for cell-cycle-related genes that could illuminate this function. IKKalpha was found to control several Myc antagonists, including Mad1, Mad2, and Ovol1, through the association with TGFbeta-regulated Smad2/3 transcription factors and is required for Smad3 recruitment to at least one of these targets. Surprisingly, Smad2/3-dependent Mad1 induction and keratinocyte differentiation are independent of Smad4, the almost universal coregulator of canonical TGFbeta signaling. IKKalpha also is needed for nuclear accumulation of activated Smad2/3 in the epidermis, and Smad2/3 are required for epidermal differentiation. We suggest that a TGFbeta-Smad2/3-IKKalpha axis is a critical Smad4-independent regulator of keratinocyte proliferation and differentiation.

Functionalization of Polycaprolactone Electrospun Osteoplastic Scaffolds with Fluorapatite and Hydroxyapatite Nanoparticles: Biocompatibility Comparison of Human Versus Mouse Mesenchymal Stem Cells.

A capability for effective tissue reparation is a living requirement for all multicellular organisms. Bone exits as a precisely orchestrated balance of bioactivities of bone forming osteoblasts and bone resorbing osteoclasts. The main feature of osteoblasts is their capability to produce massive extracellular matrix enriched with calcium phosphate minerals. Hydroxyapatite and its composites represent the most common form of bone mineral providing mechanical strength and significant osteoinductive properties. Herein, hydroxyapatite and fluorapatite functionalized composite scaffolds based on electrospun polycaprolactone have been successfully fabricated. Physicochemical properties, biocompatibility and osteoinductivity of generated matrices have been validated. Both the hydroxyapatite and fluorapatite containing polycaprolactone composite scaffolds demonstrated good biocompatibility towards mesenchymal stem cells. Moreover, the presence of both hydroxyapatite and fluorapatite nanoparticles increased scaffolds' wettability. Furthermore, incorporation of fluorapatite nanoparticles enhanced the ability of the composite scaffolds to interact and support the mesenchymal stem cells attachment to their surfaces as compared to hydroxyapatite enriched composite scaffolds. The study of osteoinductive properties showed the capacity of fluorapatite and hydroxyapatite containing composite scaffolds to potentiate the stimulation of early stages of mesenchymal stem cells' osteoblast differentiation. Therefore, polycaprolactone based composite scaffolds functionalized with fluorapatite nanoparticles generates a promising platform for future bone tissue engineering applications.

Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells.

Growth differentiation factor-9 (GDF9) is an oocyte secreted paracrine factor essential for mammalian ovarian folliculogenesis. Like other members of the transforming growth factor-beta (TGFbeta) superfamily, GDF9 is synthesized as a prepropeptide which needs processing by furin-like proteases to result in an active mature protein. We have previously characterized a preparation of unpurified recombinant mouse GDF9 which is bioactive as produced by human embryonic kidney 293T (HEK-293T) cells. However, we find that unpurified recombinant human GDF9 (hGDF9) produced by HEK-293T cells is not bioactive. Purified recombinant hGDF9 is bioactive and here we report the characterization of this protein. We find that the purified untagged mature region of hGDF9 is active in transcriptional reporter assays specific for Smad3/4 in human granulosa-luteal (hGL) cells. We also demonstrate the use of a BMP (Smad1/5) responsive (BRE-luciferase) adenovirus in primary cultures of hGL cells to detect BMP responses. Using this adenovirus we find that purified human GDF9 does not activate the Smad1/5 pathway. Purified hGDF9 mature region activated the Smad3 pathway also in the FSH responsive human granulosa tumor cell line KGN. Primary cultures of rat granulosa cells responded to purified hGDF9 with an increase in DNA synthesis as measured by [3H]-thymidine uptake. Here we also report that the inclusion of a C-terminal affinity purification tag destroys GDF9 bioactivity. This study is the first characterization of purified biologically active human GDF9 and as such is of importance for studies on human fertility, and efforts aimed at treating infertility conditions.

The Influence of Calcium Glycerophosphate (GPCa) Modifier on Physicochemical, Mechanical, and Biological Performance of Polyurethanes Applicable as Biomaterials for Bone Tissue Scaffolds Fabrication.

In this paper we describe the synthesis of poly(ester ether urethane)s (PEEURs) by using selected raw materials to reach a biocompatible polyurethane (PU) for biomedical applications. PEEURs were synthesized by using aliphatic 1,6-hexamethylene diisocyanate (HDI), poly(ethylene glycol) (PEG), α,ω-dihydroxy(ethylene-butylene adipate) (Polios), 1,4-butanediol (BDO) as a chain extender and calcium glycerolphosphate salt (GPCa) as a modifier used to stimulate bone tissue regeneration. The obtained unmodified (PURs) and modified with GPCa (PURs-M) PEEURs were studied by various techniques. It was confirmed that urethane prepolymer reacts with GPCa modifier. Further analysis of the obtained PURs and PURs-M by Fourier transform infrared (FTIR) and Raman spectroscopy revealed the chemical composition typical for PUs by the confirmed presence of urethane bonds. Moreover, the FTIR and Raman spectra indicated that GPCa was incorporated into the main PU chain at least at one-side. The scanning electron microscopy (SEM) analysis of the PURs-M surface was in good agreement with the FTIR and Raman analysis due to the fact that inclusions were observed only at 20% of its surface, which were related to the non-reacted GPCa enclosed in the PUR matrix as filler. Further studies of hydrophilicity, mechanical properties, biocompatibility, short term-interactions, and calcification study lead to the final conclusion that the obtained PURs-M may by suitable candidate material for further scaffold fabrication. Scaffolds were prepared by the solvent casting/particulate leaching technique (SC/PL) combined with thermally-induced phase separation (TIPS). Such porous scaffolds had satisfactory pore sizes (36⁻100 µm) and porosity (77⁻82%) so as to be considered as suitable templates for bone tissue regeneration.

Citrullination of epithelial neutrophil-activating peptide 78/CXCL5 results in conversion from a non-monocyte-recruiting chemokine to a monocyte-recruiting chemokine.

OBJECTIVE: To examine whether the citrullinated chemokines epithelial neutrophil-activating peptide 78 (ENA-78)/CXCL5, macrophage inflammatory protein 1α/CCL3, and monocyte chemotactic protein 1/CCL2 are detected in the biologic fluid of patients with rheumatoid arthritis (RA), and if so, to determine the biologic activities of these chemokines. METHODS: Recombinant human chemokines were citrullinated by peptidylarginine deiminase. Enzyme-linked immunosorbent assays were performed to measure the concentrations of citrullinated chemokines in sera from patients with rheumatoid arthritis (RA) and normal individuals and in synovial fluid from patients with RA, patients with osteoarthritis (OA), and patients with other inflammatory rheumatic diseases. The correlation between the citrullinated chemokine levels and clinical data was analyzed. Monocyte and neutrophil chemotaxis assays were performed, and native (noncitrullinated) or citrullinated ENA-78/CXCL5 was injected into mouse knees to evaluate the biologic activities of these chemokines. RESULTS: The concentration of citrullinated ENA-78/CXCL5 was significantly higher in RA sera and SF than in normal sera and in SF from patients with other rheumatic diseases including OA. In RA SF, a strong correlation between the amount of citrullinated ENA-78/CXCL5 and the C-reactive protein level or the erythrocyte sedimentation rate was observed. Citrullinated ENA-78/CXCL5 induced monocyte chemotaxis via CXCR1 and CXCR2, while noncitrullinated ENA-78/CXCL5 did not. In a mouse model of inflammatory arthritis, citrullinated ENA-78/CXCL5 induced more severe inflammation and recruited more monocytes than did noncitrullinated ENA-78/CXCL5. CONCLUSION: Citrullinated ENA-78/CXCL5 is highly correlated with RA disease activity and, unlike noncitrullinated ENA-78/CXCL5, recruits monocytes. These results indicate that citrullinated ENA-78/CXCL5 may exert previously unrecognized inflammatory properties in RA by recruiting monocytes to inflamed joint tissue.

The Rho-GEF Trio regulates a novel pro-inflammatory pathway through the transcription factor Ets2.

Inflammation is characterized by endothelium that highly expresses numerous adhesion molecules to trigger leukocyte extravasation. Central to this event is increased gene transcription. Small Rho-GTPases not only control the actin cytoskeleton, but are also implicated in gene regulation. However, in inflammation, it is not clear how this is regulated. Here, we show that the guanine-nucleotide exchange factor Trio expression is increased upon inflammatory stimuli in endothelium. Additionally, increased Trio expression was found in the vessel wall of rheumatoid arthritis patients. Trio silencing impaired VCAM-1 expression. Finally, we excluded that Trio-controlled VCAM-1 expression used the classical NFκB or MAP-kinase pathways, but rather acts on the transcriptional level by increasing phosphorylation and nuclear translocalization of Ets2. These data implicate Trio in regulating inflammation and provide novel targets for therapeutic purposes to treat inflammatory diseases such as rheumatoid arthritis.

Biphasic effects of transforming growth factor ß on bone morphogenetic protein-induced osteoblast differentiation.

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP-induced bone formation may lead to the development of more effective BMP-based therapies. To identify kinases mediating BMP-induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP-6-induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor ß (TGF-ß) generally is considered to antagonize BMP-induced osteoblast differentiation, C2C12 cells transfected with siRNAs targeting TGF-ß receptors displayed reduced BMP-6-induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF-ß type I receptor impaired BMP-induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF-ß further increased expression of osteoblast-specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF-ß were found to depend on timing and duration of the costimulation. TGF-ß inhibited BMP-induced activation of a BMP-Smad-dependent luciferase reporter, suggesting that the stimulatory effect of TGF-ß is not due to increased BMP-Smad activity. TGF-ß also inhibited the BMP-induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF-ß, besides acting as an inhibitor, also can, by dampening the noggin-mediated negative-feedback loop, enhance BMP-induced osteoblast differentiation, which might be beneficial in fracture healing.

The bioactivity of human bone morphogenetic protein-15 is sensitive to C-terminal modification: characterization of the purified untagged processed mature region.

Oocyte-derived bone morphogenetic protein-15 (BMP15) is critical for the regulation of mammalian fertility. Previously we have found that a C-terminal His(6)-tag destroys the bioactivity of growth differentiation-9 (GDF9, a homolog of BMP15). In this study we found that recombinant human BMP15 is produced by HEK-293T cells in an active form, but the bioactivity is lost by C-terminal modification, specifically, fusion to a Flag tag. After purification the mature BMP15 wt is active in transcriptional reporter assays specific for Smad1/5/8 in human granulosa-luteal (hGL) and COV434 granulosa tumor cells, whereas BMP15 with a carboxy-terminal Flag tag remains inactive. Using these same cell models we found that treatment with purified mature BMP15 wt causes a rapid phosphorylation of Smad1. The purified BMP15 wt is a potent stimulator of rat granulosa cell DNA synthesis, which could be antagonized by the BMPRII ectodomain-Fc fusion molecule, whereas the BMP15C-Flag was completely inactive. Further, the BMP15 wt form is a potent stimulator of inhibin B production in hGL cells. We found that the purified BMP15 wt consists of P16 and -17, both of which are post-translationally modified forms. This is the first characterization of a purified untagged human BMP15 mature region, which is stable and highly bioactive in human and rodent granulosa cells and as such is of importance for studies on human fertility.