Methylation of the epigenetic JMJD2D protein by SET7/9 promotes prostate tumorigenesis.

How the function of the JMJD2D epigenetic regulator is regulated or whether it plays a role in prostate cancer has remained elusive. We found that JMJD2D was overexpressed in prostate tumors, stimulated prostate cancer cell growth and became methylated by SET7/9 on K427. Mutation of this lysine residue in JMJD2D reduced the ability of DU145 prostate cancer cells to grow, invade and form tumors and elicited extensive transcriptomic changes. This included downregulation of CBLC, a ubiquitin ligase gene with hitherto unknown functions in prostate cancer, and upregulation of PLAGL1, a transcription factor with reported tumor suppressive characteristics in the prostate. Bioinformatic analyses indicated that CBLC expression was elevated in prostate tumors. Further, downregulation of CBLC largely phenocopied the effects of the K427 mutation on DU145 cells. In sum, these data have unveiled a novel mode of regulation of JMJD2D through lysine methylation, illustrated how this can affect oncogenic properties by influencing expression of the CBLC gene, and established a pro-tumorigenic role for CBLC in the prostate. A corollary is that JMJD2D and CBLC inhibitors could have therapeutic benefits in the treatment of prostate and possibly other cancers.

SET7/9-mediated methylation affects oncogenic functions of histone demethylase JMJD2A.

The histone demethylase JMJD2A/KDM4A facilitates prostate cancer development, yet how JMJD2A function is regulated has remained elusive. Here, we demonstrate that SET7/9-mediated methylation on 6 lysine residues modulated JMJD2A. Joint mutation of these lysine residues suppressed JMJD2A's ability to stimulate the MMP1 matrix metallopeptidase promoter upon recruitment by the ETV1 transcription factor. Mutation of just 3 methylation sites (K505, K506, and K507) to arginine residues (3xR mutation) was sufficient to maximally reduce JMJD2A transcriptional activity and also decreased its binding to ETV1. Introduction of the 3xR mutation into DU145 prostate cancer cells reduced in vitro growth and invasion and also severely compromised tumorigenesis. Consistently, the 3xR genotype caused transcriptome changes related to cell proliferation and invasion pathways, including downregulation of MMP1 and the NPM3 nucleophosmin/nucleoplasmin gene. NPM3 downregulation phenocopied and its overexpression rescued, to a large degree, the 3xR mutation in DU145 cells, suggesting that NPM3 was a seminal downstream effector of methylated JMJD2A. Moreover, we found that NPM3 was overexpressed in prostate cancer and might be indicative of disease aggressiveness. SET7/9-mediated lysine methylation of JMJD2A may aggravate prostate tumorigenesis in a manner dependent on NPM3, implying that the SET7/9→JMJD2A→NPM3 axis could be targeted for therapy.

IRE1α arm of unfolded protein response in muscle-specific TGF-ß signaling-mediated regulation of muscle cell immunological properties.

Endoplasmic reticulum stress (ERS) and the unfolded protein response (UPR) are involved in various muscle pathological states. The IRE1α arm of UPR can affect immunological properties of myofiber through restraining p38 mitogen-activated protein kinases (MAPK) activation under inflammatory milieu. However, the relevant pathway molecules regulating the initiation of the IRE1α arm in myofiber remain unclear. In this work, expression of transforming growth factor-beta (TGF-ß) and TGF-ß receptor II (TGF-ßr2), and UPR pathway activation were examined in cardiotoxin (CTX)-damaged mouse muscle, which revealed the activation of TGF-ß signaling and UPR in CTX-damaged muscle and in regenerating myofibers. Using control or transgenic mice with TGF-ßr2 deleted in skeletal muscle (SM TGF-ßr2-/-) and the derived primary differentiating myogenic precursor cells (MPCs) treated with/without ERS activator or inhibitor, IRE1α pathway inhibitor, or TGF-ß signaling activator, this study further revealed an essential role of intrinsic TGF-ß signaling in regulating muscle cell to express inflammation-related molecules including H-2Kb, H2-Eα, TLR3, and special myokines. TGF-ß signaling prompted UPR IRE1α arm and restrained p38 MAPK activation in myofiber under inflammatory milieu. This study uncovers a previously unrecognized function of TGF-ß signaling acting as an upstream factor controlling myofiber immune capacities in the inflamed state through the UPR-IRE1α-p38 MAPK pathway.

Methylation of the JMJD2B epigenetic regulator differentially affects its ability to coactivate the ETV1 and JUN transcription factors.

OBJECTIVES: Jumonji C domain-containing (JMJD) 2B (JMJD2B) is a transcriptional cofactor and histone demethylase that is involved in prostate cancer formation. However, how its function is regulated by posttranslational modification has remained elusive. Hence, we examined if JMJD2B would be regulated by lysine methylation. METHODS: Through in vitro methylation assays and Western blotting with methyl-lysine specific antibodies, we analyzed lysine methylation within JMJD2B. Identified methylated lysine residues were mutated to arginine residues and the respective impact on JMJD2B transcriptional activity measured with a reporter gene assay in human LNCaP prostate cancer cells. RESULTS: We discovered that JMJD2B is methylated on up to six different lysine residues. Further, we identified the suppressor of variegation 3-9/enhancer of zeste/trithorax (SET) domain-containing protein 7/9 (SET7/9) as the methyltransferase being responsible for this posttranslational modification. Mutating the methylation sites in JMJD2B to arginine residues led to diminished coactivation of the Ju-nana (JUN) transcription factor, which is a known oncogenic protein in prostate tumors. In contrast, methylation of JMJD2B had no impact on its ability to coactivate another transcription factor associated with prostate cancer, the DNA-binding protein E26 transformation-specific (ETS) variant 1 (ETV1). Consistent with a potential joint action of JMJD2B, SET7/9 and JUN in prostate cancer, the expression of JMJD2B in human prostate tumors was positively correlated with both SET7/9 and JUN levels. CONCLUSIONS: The identified SET7/9-mediated methylation of JMJD2B appears to impact its cooperation with selected interacting transcription factors in prostate cancer cells. Given the implicated roles of JMJD2B beyond prostate tumorigenesis, SET7/9-mediated methylation of JMJD2B possibly also influences the development of other cancers, while its impairment might have relevance for obesity or a global developmental delay that can be elicited by reduced JMJD2B activity.

Regenerating myofiber directs Tregs and Th17 responses in inflamed muscle through the intrinsic TGF-ß signaling-mediated IL-6 production.

Transforming growth factor-ß (TGF-ß) is considered to be an important immune regulatory cytokine. However, it remains unknown whether and how the muscle fiber specific-TGF-ß signaling is directly involved in intramuscular inflammatory regulation by affecting T cells. Here, we addressed these in a mouse tibialis anterior muscle Cardiotoxin injection-induced injury repair model in muscle creatine kinase (MCK)-Cre control or transgenic mice with TGF-ß receptor II (TGF-ßr2) being specifically deleted in muscle cells (SM TGF-ßr2-/-). In control mice, TGF-ß2 and TGF-ßr2 were found significantly upregulated in muscle after the acute injury. In mutant mice, deficiency of TGF-ß signaling in muscle cells caused more serious muscle inflammation, with the increased infiltration of macrophages and CD4+ T cells at the degeneration stage (D4) and the early stage of regeneration (D7) after myoinjury. Notably, the loss of TGF-ß signaling in myofibers dramatically affected CD4+ T cell function and delayed T cells withdrawal at the later stage of muscle regeneration (D10 and D15), marked by the elevated Th17, but the impaired Tregs response. Furthermore, in vivo and in vitro, the intrinsic TGF-ß signaling affected immune behaviors of muscle cells and directed CD4+ T cells differentiation by impairing IL-6 production and release. It suggests that local muscle inflammation can be inhibited potentially by directly activating the TGF-ß signaling pathway in muscle cells to suppress Th17, but induce Tregs responses. Thus, according to the results of this study, we found a new idea for the control of local acute inflammation in skeletal muscle.NEW & NOTEWORTHY Myofiber mediates muscle inflammatory response through activating the intrinsic TGF-ß signaling. The specific TGF-ß signaling activation contributes to myofiber IL-6 production and directs muscle-specific Th17 and Treg cell responses.

Crucial Functions of the JMJD1/KDM3 Epigenetic Regulators in Cancer.

Epigenetic changes are one underlying cause for cancer development and often due to dysregulation of enzymes modifying DNA or histones. Most Jumonji C domain-containing (JMJD) proteins are histone lysine demethylases (KDM) and therefore epigenetic regulators. One JMJD subfamily consists of JMJD1A/KDM3A, JMJD1B/KDM3B, and JMJD1C/KDM3C that are roughly 50% identical at the amino acid level. All three JMJD1 proteins are capable of removing dimethyl and monomethyl marks from lysine 9 on histone H3 and might also demethylate histone H4 on arginine 3 and nonhistone proteins. Analysis of knockout mice revealed critical roles for JMJD1 proteins in fertility, obesity, metabolic syndrome, and heart disease. Importantly, a plethora of studies demonstrated that especially JMJD1A and JMJD1C are overexpressed in various tumors, stimulate cancer cell proliferation and invasion, and facilitate efficient tumor growth. However, JMJD1A may also inhibit the formation of germ cell tumors. Likewise, JMJD1B appears to be a tumor suppressor in acute myeloid leukemia, but a tumor promoter in other cancers. Notably, by reducing methylation levels on histone H3 lysine 9, JMJD1 proteins can profoundly alter the transcriptome and thereby affect tumorigenesis, including through upregulating oncogenes such as CCND1, JUN, and MYC This epigenetic activity of JMJD1 proteins is sensitive to heavy metals, oncometabolites, oxygen, and reactive oxygen species, whose levels are frequently altered within cancer cells. In conclusion, inhibition of JMJD1 enzymatic activity through small molecules is predicted to be beneficial in many different cancers, but not in the few malignancies where JMJD1 proteins apparently exert tumor-suppressive functions.

The Thickness of Surface Grafting Layer on Bio-materials directly Mediates the Immuno-reacitivity of Macrophages in vitro.

Introducing the surface grafting layers to regulate bio-compatibility and bio-function is an important step in the tissue engineering field. However, whether the thickness variation of the introduced biomimetic layer impacts the behavior of the adhered immune effector cells is yet to be dissected fully. In this study, we used a surface-induced atom transfer radical polymerization (SI-ATRP) method to synthetize and graft poly-phenoxyethyl methacrylate (PHEMA) brushes having different lengths on the glass substrates. Primary murine peritoneal macrophages were collected and cultured on the PHEMA brushes and we investigated the influence of polymer brushes having different lengths on macrophages phenotype and function. Our results demonstrated that the thicker brushes (200 nm and 450 nm) are superior to the thinner layers (50 nm) for macrophages survival, proliferation, cell elongation and migration. Moreover, the thicker brushes are more beneficial for macrophage's activities and functions, presented by the increased production of M1-associated cytokines IL-6 and MCP-1, the elevated cell phagocytosis and the activation molecule F4/80 expression, and the reduced macrophages apoptosis in thicker brushes-sustained macrophages. Our data suggests that the thickness of the substrate grafting layer directly impacts macrophages recruitment and pro-inflammatory function, which is important in determining the intrinsic immuno-compatibilities of the surface modified-biomaterials and mediates material-host interactions in vivo.

The IRE1α Arm of UPR Regulates Muscle Cells Immune Characters by Restraining p38 MAPK Activation.

Skeletal muscle repair and systemic inflammation/immune responses are linked to endoplasmic reticulum stress (ER stress) pathways in myopathic muscle, and muscle cells play an active role in muscular immune reactions by exhibiting immunological characteristics under persistent proinflammation stimuli. Whether ER stress affects the intrinsic immunological capacities of myocytes in the inflammatory milieu, as it does to immune cells, and which arms of the unfolded protein response (UPR) mainly participate in these processes remain mostly unknown. We investigated this issue and showed that inflammatory stimuli can induce the activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1α (IRE1α) arms of the UPR in myocytes both in vivo and in vitro. UPR stressor administration reversed the increased IFN-γ-induced expression of the MHC-II molecule H2-Ea, the MHC-I molecule H-2K b , toll-like receptor 3 (TLR3) and some proinflammatory myokines in differentiated primary myotubes in vitro. However, further IRE1α inhibition thoroughly corrected the trend in the UPR stressor-triggered suppression of immunobiological molecules. In IFN-γ-treated myotubes, dramatic p38 MAPK activation was observed under IRE1α inhibitory conditions, and the pharmacological inhibition of p38 reversed the immune molecule upregulation induced by IRE1α inhibition. In parallel, our coculturing system verified that the ovalbumin (OVA) antigen presentation ability of inflamed myotubes to OT-I T cells was enhanced by IRE1α inhibition, but was attenuated by further p38 inhibition. Thus, the present findings demonstrated that p38 MAPK contributes greatly to IRE1α arm-dependent immunobiological suppression in myocytes under inflammatory stress conditions.

Calmodulin-dependent signalling pathways are activated and mediate the acute inflammatory response of injured skeletal muscle.

KEY POINTS: There is a close relationship between skeletal muscle physiology and Ca2+ /calmodulin (CaM) signalling. Despite the effects of Ca2+ /CaM signalling on immune and inflammatory responses having been extensively explored, few studies have investigated the role of CaM pathway activation on the post-injury muscle inflammatory response. In this study, we investigated the role of CaM-dependent signalling in muscle inflammation in cardiotoxin induced myoinjuries in mice. The Ca2+ /calmodulin-dependent protein kinase II (CaMII), Ca2+ /calmodulin-dependent protein kinase IV (CaMKIV), and nuclear factor of activated T cells (NFAT) pathways are likely to be simultaneously activated in muscle cells and in infiltrating lymphocytes and to regulate the immune behaviours of myofibres in an inflammatory environment, and these pathways ultimately affect the outcome of muscle inflammation. ABSTRACT: Calcium/calmodulin (Ca2+ /CaM) signalling is essential for immune and inflammatory responses in tissues. However, it is unclear if Ca2+ /CaM signalling interferes with muscle inflammation. Here we investigated the roles of CaM-dependent signalling in muscle inflammation in mice that had acute myoinjuries in the tibialis anterior muscle induced by intramuscular cardiotoxin (CTX) injections and received intraperitoneal injections of either the CaM inhibitor calmidazolium chloride (CCL) or CaM agonist calcium-like peptide 1 (CALP1). Multiple inflammatory parameters, including muscle autoantigens and toll-like receptors, mononuclear cell infiltration, cytokines and chemokines associated with peripheral muscle inflammation, were examined after the injury and treatment. CALP1 treatment enhanced intramuscular infiltration of monocytes/macrophages into the damaged tibialis anterior muscle and up-regulated mRNA and protein levels of muscle autoantigens (Mi-2, HARS and Ku70) and Toll-like receptor 3 (TLR3), and mRNA levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), Monocyte chemoattractant protein-1 (MCP1), Monocyte chemoattractant protein-3 (MCP3) and Macrophage inflammatory protein-1(MIP-1α) in damaged muscle. In contrast, CCL treatment decreased the intramuscular cell infiltration and mRNA levels of the inflammatory mediators. After CALP1 treatment, a substantial up-regulation in Ca2+ /calmodulin-dependent protein kinase II (CaMKII), Ca2+ /calmodulin-dependent protein kinase IV (CaMKIV) and nuclear factor of activated T cells (NFAT) activity was detected in CD45+ cells isolated from the damaged muscle. More pro-inflammatory F4/80+ Ly-6C+ cells were detected in CD45-gated cells after CALP1 treatment than in those after CCL treatment or no treatment. Consistently, in interferon-γ-stimulated cultured myoblasts and myotubes, CALP1 treatment up-regulated the activities of CaMKII, CaMKIV and NFAT, and levels of class I/II major histocompatibility complexes (MHC-I/II) and TLR3. Our findings demonstrated that CaM-dependent signalling pathways mediate the injury-induced acute muscle inflammatory response.

The thickness of poly-phenoxyethyl methacrylate brush interferes with cellular behavior and function of myofibers.

Introducing or grafting molecules onto biomaterial surfaces to regulate muscle cell destination via biophysical cues is one of the important steps for biomaterial design in muscle tissue engineering. Therefore, it is important to understand the interaction between myoblasts and myofibers with substrates modified by biomimetic layer with different thicknesses. In this study, we used a surface-induced atom transfer radical polymerization method to synthetize and graft poly-phenoxyethyl methacrylate (PHEMA) brushes having different lengths on the glass substrates. C2C12 myoblasts were seeded on the PHEMA brushes and differentiated using horse serum, for analyzing the sensibility of muscle cells to feel environment changing, and further investigating whether the depths of grafting layer on the biomaterial surface are important factors in regulating muscle cell behaviors. Our results demonstrated that on the thicker PHEMA brushes surface (200 and 450 nm), C2C12 myoblasts showed a better survival and proliferation and were favorable for cell fusion and myotube formation. Furthermore, myofibers survived on the thicker brushes were more functional and upregulated cytoskeleton proteins (tubulin, vimentin, and vinculin) and FAK levels, and enhanced the expression levels for mechanical stress molecules (HGF, NOS-1, and c-Met). These results suggest that grafting thickness of PHEMA layer on the substrate led to the myoblasts/myofiber behavior change, which would be valuable for the design and preparation of the modified layer on muscle tissue engineering scaffolds. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1264-1272, 2019.

Immunological Behavior Analysis of Muscle Cells under IFN-γ Stimulation in Vitro and in Vivo.

Muscle cells could serve as antigen-presenting cells, and participate in the activation of immune response. Immunological characteristics of muscle cells, and their capacities to equip themselves with immunorelevant molecules, remain to be elucidated. In this study, we investigated the immunological properties of myoblasts and differentiated myotubes in vitro and in vivo, under the IFN-γ induced inflammatory condition. We found that the fused C2 C12 myotubes are more sensitive to inflammatory stimulation, and significantly upregulated the expression levels of MHC-I/II and TLR3/7 molecules, than that of proliferated myoblasts. As well, some co-stimulatory/-inhibitory molecules, including CD40, CD86, ICAM-I, ICOS-L, and PD-L1, were prominently upregulated in IFN-γ induced myotubes. Notably, we detected the protein levels of ASC, NLRP3, and Caspase-1 increased in stimulated myotubes, and IL-1ß in cell culture supernatant, implying the activation of NLRP3 inflammasomes in IFN-γ treated myotubes. The pro-inflammatory cytokines and chemokines mRNA levels in IFN-γ induced C2 C12 myotubes and myoblasts, involving IL-1, IL-6, and MCP-1, increased markedly. T cell activation test further verified IFN-γ induced C2 C12 myotubes prompt to the proliferation of the splenic CD4+ and CD8+ T cells. In Cardiotoxin-damaged tibialis anterior (TA) muscle, some regenerated myofibers expressed both MHC class I and class II molecules under IFN-γ enhanced inflammatory condition. Thus, our work demonstrates that muscle cells are active participants of local immune reactions. Anat Rec, 301:1551-1563, 2018. © 2018 Wiley Periodicals, Inc.

Calcium/Calmodulin-Dependent Protein Kinase IV Mediates IFN-γ-Induced Immune Behaviors in Skeletal Muscle Cells.

BACKGROUND/AIMS: Whether calcium/calmodulin-dependent protein kinase IV (CaMKIV) plays a role in regulating immunologic features of muscle cells in inflammatory environment, as it does for immune cells, remains mostly unknown. In this study, we investigated the influence of endogenous CaMKIV on the immunological characteristics of myoblasts and myotubes received IFN-γ stimulation. METHODS: C2C12 and murine myogenic precursor cells (MPCs) were cultured and differentiated in vitro, in the presence of pro-inflammatory IFN-γ. CaMKIV shRNA lentivirus transfection was performed to knockdown CaMKIV gene in C2C12 cells. pEGFP-N1-CaMKIV plasmid was delivered into knockout cells for recovering intracellular CaMKIV gene level. CREB1 antagonist KG-501 was used to block CREB signal. qPCR, immunoblot analysis, or immunofluorescence was used to detect mRNA and protein levels of CaMKIV, immuno-molecules, or pro-inflammatory cytokines and chemokines. Co-stimulatory molecules expression was assessed by FACS analysis. RESULTS: IFN-γ induces the expression or up-regulation of MHC-I/II and TLR3, and the up-regulation of CaMKIV level in muscle cells. In contrast, CaMKIV knockdown in myoblasts and myotubes leads to expression inhibition of the above immuno-molecules. As well, CaMKIV knockdown selectively inhibits pro-inflammatory cytokines/chemokines, and co-stimulatory molecules expression in IFN-γ treated myoblasts and myotubes. Finally, CaMKIV knockdown abolishes IFN-γ induced CREB pathway molecules accumulation in differentiated myotubes. CONCLUSIONS: CaMKIV can be induced to up-regulate in muscle cells under inflammatory condition, and positively mediates intrinsic immune behaviors of muscle cells triggered by IFN-γ.

In vivo immuno-reactivity analysis of the porous three-dimensional chitosan/SiO2 and chitosan/SiO2 /hydroxyapatite hybrids.

Inorganic/organic hybrid silica-chitosan (CS) scaffolds have promising potential for bone defect repair, due to the controllable mechanical properties, degradation behavior, and scaffold morphology. However, the precise in vivo immuno-reactivity of silica-CS hybrids with various compositions is still poorly defined. In this study, we fabricated the three-dimensional (3D) interconnected porous chitosan-silica (CS/SiO2 ) and chitosan-silica-hydroxyapatite (CS/SiO2 /HA) hybrids, through sol-gel process and 3D plotting skill, followed by the naturally or freeze drying separately. Scanning electron microscopy demonstrated the hybrids possessed the uniform geometric structure, while, transmission electron microscopy displayed nanoscale silica, or HA nanoparticles dispersed homogeneously in the CS matrix, or CS/silica hybrids. After intramuscular implantation, CS/SiO2 and CS/SiO2 /HA hybrids triggered a local and limited monocyte/macrophage infiltration and myofiber degeneration. Naturally dried CS/SiO2 hybrid provoked a more severe inflammation than the freeze-dried ones. Dendritic cells were attracted to invade into the implants embedded-muscle, but not be activated to prime the adaptive immunity, because the absence of cytotoxic T cells and B cells in muscle received the implants. Fluorescence-activated cell sorting (FACS) analysis indicated the implanted hybrids were incapable to initiate splenocytes activation. Plasma complement C3 enzyme linked immunosorbent assay (ELISA) assay showed the hybrids induced C3 levels increase in early implanting phase, and the subsequent striking decrease. Thus, the present results suggest that, in vivo, 3D plotted porous CS/SiO2 and CS/SiO2 /HA hybrids are relatively biocompatible in vivo, which initiate a localized inflammatory procedure, instead of a systematic immune response. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1223-1235, 2018.

Calcium/Calmodulin-Dependent Protein Kinase IV (CaMKIV) Mediates Acute Skeletal Muscle Inflammatory Response.

The objective of this study is to investigate the role of Calmodulin-dependent protein kinase IV (CaMKIV) in Cardiotoxin (CTX)-induced mice muscle inflammation. CTX injection i.m. was performed to induce B6 mice acute tibialis anterior (TA) muscle injury. The mice were then injected i.p. with the recombinant CaMKIV protein or its antagonist KN-93. Immunoblotting was used to assess Calmodulin (CaM) and CaMKIV levels. Immunofluorescence was used to detect intramuscular infiltration or major histocompatibility complex (MHC)-I expression in damaged muscle. The extent of infiltration was evaluated by fluorescent intensity analysis. Cytokines/chemokines levels were determined by qPCR. CaMKIV gene knockdown in C2C12 cells was performed in order to evaluate the effects of CaMKIV on immuno-behavior of muscle cells. CTX administration induced a strong up-regulation of CaM and p-CaMKIV levels in infiltrated mononuclear cells and regenerated myofibers. In vivo adding of the recombinant CaMKIV protein enhanced intramuscular infiltration of monocytes/macrophages in damaged muscle and increased the number of proinflammatory Ly-6C+F4/80+ macrophage cells. CaMKIV protein treatment induced a striking up-regulation of mRNA levels of IL-1, IL-6, MCP-1, and MCP-3 in CD45+ cells sorted from damaged muscle; increased the infiltration of CD8+ T cells; and induced the up-regulation of MHC-I in partial regenerated myofibers, which was rarely observed in muscle damage alone. Additionally, CaMKIV protein treatment diminished the regulatory T cells (Tregs) number and led to the damaged TA muscle repair delay. In vitro CaMKIV gene knockdown reversed IFN-γ-induced up-regulation of MHC-I/II and TLR3 in the differentiated C2C12 myotubes. CaMKIV can act as an immunostimulation molecule and enhances the acute muscle inflammatory responses.

Polyurethane conjugating TGF-ß on surface impacts local inflammation and endoplasmic reticulum stress in skeletal muscle.

The synthesized short peptide-polymers would provide key functions for tissue regeneration and repair, through enriching bioactive molecules on polymers or releasing these molecules pre-conjugated on the materials. We have developed a degradable polyurethane (PU) bearing HSNGLPL peptide, which has affinity binding ability to transforming growth factor-betas (TGF-ß). For deeply understanding spatial release of TGF-ß from the PU polymers and its localized bioactivity, quartz crystal microbalance (QCM) and Elisa test were used to verify TGF-ß binding capacities in vitro and in vivo. The PU polymers, with or without pre-conjugating of TGF-ß, were implanted into gastronomies muscle (GN) of C57BL/6 mice, for addressing TGF-ß release from the polymers and its bio-regulating function in vivo. QCM result shows that PU bearing HSNGLPL peptide has affinity binding ability to TGF-ß in vitro. Intramuscular implanting experiment further supports the enrichment efficiency of TGF-ß on PU polymers in vivo. The detecting data involving intramuscular inflammatory infiltration triggered by the implants, myofiber regeneration, muscular fibrosis degree, and activation of endoplasmic reticulum stress (ER stress), evidence TGF-ß can be released from PU polymers, and exerts regulating effects on the material-induced inflammation. Thus, our present results suggest it is feasible to improve biocompatibility of PU polymers in vivo, by pre-bearing bioactive molecules on materials before the implanting. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1156-1165, 2017.

Inflammatory and immuno-reactivity in mice induced by intramuscular implants of HSNGLPL peptide grafted-polyurethane.

Synthetic peptide-based polyurethanes (PUs), introduced as bioactive agents and possessing impressive properties, have emerged as attractive functional biomaterials for tissue regeneration. In this study, we developed a PU with a pendent HSNGLPL group through click reaction, which has strong affinity to TGF-ß1. The peptide grafted-PUs, or PUs with BDO as the chain extender (control), were implanted into the gastrocnemius muscle (GN) of C57BL/6 mice, for evaluating their inflammatory and immuno-reactivity in vivo. We show herein that, after muscle implantation, BDO-PU induced a conspicuous monocyte/macrophage infiltration and myofiber degeneration. The inflammatory invasion and myofiber necrosis were mainly detected in the site around, but not far from, the implants, suggesting that the degraded PU matrix only triggers a local and limited inflammation in vivo. In contrast, peptide grafted-PU induced intramuscular inflammation was more complex and was sustained for more than 2 months. Apart from nonspecific monocyte/macrophage infiltration as in the case of BDO-PU, CD4+ T cells and dendritic cells (DCs), the members of the adaptive immune system, can be detected within the inflammatory site around peptide grafted-PU implants. The number of apoptotic macrophages in muscle containing peptide-PU was significantly lower compared to that in muscle containing BDO-PU. Thus, our present results suggest that, the PU matrix degradation-produced local environment is toxic to muscle cells and induces muscle degeneration. Moreover, highly aggregated peptide on PU might act as an immunogen to trigger intramuscular inflammation and lead to the delayed inflammatory response.