MD1 deletion exaggerates cardiomyocyte autophagy induced by heart failure with preserved ejection fraction through ROS/MAPK signalling pathway.

In our previous studies, we reported that myeloid differentiation protein 1 (MD1) serves as a negative regulator in several cardiovascular diseases. However, the role of MD1 in heart failure with preserved ejection fraction (HFpEF) and the underlying mechanisms of its action remain unclear. Eight-week-old MD1-knockout (MD1-KO) and wild-type (WT) mice served as models of HFpEF induced by uninephrectomy, continuous saline or d-aldosterone infusion and a 1.0% sodium chloride treatment in drinking water for 4 weeks to investigate the effect of MD1 on HFpEF in vivo. H9C2 cells were treated with aldosterone to evaluate the role of MD1 KO in vitro. MD1 expression was down-regulated in the HFpEF mice; HFpEF significantly increased the levels of intracellular reactive oxygen species (ROS) and promoted autophagy; and in the MD1-KO mice, the HFpEF-induced intracellular ROS and autophagy effects were significantly exacerbated. Moreover, MD1 loss activated the p38-MAPK pathway both in vivo and in vitro. Aldosterone-mediated cardiomyocyte autophagy was significantly inhibited in cells pre-treated with the ROS scavenger N-acetylcysteine (NAC) or p38 inhibitor SB203580. Furthermore, inhibition with the autophagy inhibitor 3-methyladenine (3-MA) offset the aggravating effect of aldosterone-induced autophagy in the MD1-KO mice and cells both in vivo and in vitro. Our results validate a critical role of MD1 in the pathogenesis of HFpEF. MD1 deletion exaggerates cardiomyocyte autophagy in HFpEF via the activation of the ROS-mediated MAPK signalling pathway.

Two-photon fluorescence lifetime imaging of intrinsic NADH in three-dimensional tumor models.

Most studies using intrinsic NAD(P)H as biomarkers for energy metabolism and mitochondrial anomalies have been conducted in routine two-dimensional (2D) cell culture formats. Cellular metabolism and cell behavior, however, can be significantly different in 2D cultures from that in vivo. As a result, there are emerging interests in integrating noninvasive, quantitative imaging techniques of NAD(P)H with in vivo-like three-dimensional (3D) models. The overall features and metabolic responses of the murine breast cancer cells line 4T1 in 2D cultures were compared with those in 3D collagen matrix using integrated optical micro-spectroscopy. The metabolic responses to two novel compounds, MD1 and TPPBr, that target metabolism by disrupting monocarboxylate transporters or oxidative phosphorylation (OXPHOS), respectively, were investigated using two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intracellular NAD(P)H in 2D and 3D cultures. 4T1 cells exhibit distinct behaviors in a collagenous 3D matrix from those in 2D culture, forming anastomosing multicellular networks and spherical acini in 3D culture, as opposed to simple flattened epithelial plaques in 2D culture. The cellular NAD(P)H in 3D collagen matrix exhibits a longer fluorescence lifetime as compared with 2D culture, which is attributed to an enhanced population of enzyme-bound NAD(P)H in the 3D culture. TPPBr induces mitochondrial hyperpolarization in 2D culture of 4T1 cells along with an enhanced free NAD(P)H population, which suggest an interference with OXPHOS. In contrast, 2P-FLIM of cellular NAD(P)H revealed an enhanced autofluorescence lifetime in 3D 4T1 cultures after MD1 treatment as compared with MD1-treated 2D culture and the control 3D culture. Physical and chemical microenvironmental signaling are critical factors in understanding how therapeutic compounds target cancer cells by disrupting their metabolic pathways. Integrating 2P-FLIM of intrinsic NAD(P)H with refined 3D tumor-matrix in vitro models promises to advance our understanding of the roles of metabolism and metabolic plasticity in tumor growth and metastatic behavior. © 2018 International Society for Advancement of Cytometry.

Inflammatory responses increase secretion of MD-1 protein.

Radioprotective 105 (RP105) is a type I transmembrane protein, which associates with a glycoprotein, MD-1. Monoclonal antibody (mAb)-mediated ligation of RP105/MD-1 robustly activates B cells. RP105/MD-1 is structurally similar to Toll-like receptor 4 (TLR4)/MD-2. B-cell responses to TLR2 and TLR4/MD-2 ligands are impaired in the absence of RP105 or MD-1. In addition to RP105/MD-1, MD-1 alone is secreted. The structure of MD-1 shows that MD-1 has a hydrophobic cavity that directly binds to phospholipids. Little is known, however, about a ligand for MD-1 and the role of MD-1 in vivo To study the role of RP105/MD-1 and MD-1 alone, specific mAbs against MD-1 are needed. Here, we report the establishment and characterization of two anti-MD-1 mAbs (JR2G9, JR7G1). JR2G9 detects soluble MD-1, whereas JR7G1 binds both soluble MD-1 and the cell surface RP105/MD-1 complex. With these mAbs, soluble MD-1 was detected in the serum and urine. The MD-1 concentration was altered by infection, diet and reperfusion injury. Serum MD-1 was rapidly elevated by TLR ligand injection in mice. The quantitative PCR and supernatant-precipitated data indicate that macrophages are one of the sources of serum soluble MD-1. These results suggest that soluble MD-1 is a valuable biomarker for inflammatory diseases.

Molecular dysfunctions in acute rejection after renal transplantation revealed by integrated analysis of transcription factor, microRNA and long noncoding RNA.

Acute rejection remains a problem in renal transplantation. To further illustrate the mechanism of rejection, we integrated protein array-based proteomics and RNA microarray-based genomics to investigate the transcription factor, microRNA and long noncoding RNA of biopsies of three patients with acute rejections and a control group. 99 transcription factors were identified in acute rejection biopsies compared to normal renal tissue. We correlated transcription factor data with microRNA and long noncoding RNA data sets and reported the expression of 5 transcription factors (AP-1, AP-4, STATx, c-Myc and p53), 12 miRNAs and 32 lncRNAs in acute rejection biopsies. Pathway analysis demonstrated that over-presentation of transcription factor pathway plays a critical role in acute rejection. This is the first study to comprehensively report the acute rejection transcription factor pathway. Integrative analysis of the transcription factor, microRNA and long noncoding RNA provided an expansive view of molecular signaling pathways in acute rejection after renal transplantation.

MD-1 downregulation is associated with reduced cell surface CD180 expression in CLL.

CD180 is a toll-like receptor that is highly expressed in complex with the MD-1 satellite molecule on the surface of B cells. In chronic lymphocytic leukaemia (CLL) however, the expression of CD180 is highly variable and overall, significantly reduced when compared to normal B cells. We have recently shown that reduced CD180 expression in CLL lymph nodes is associated with inferior overall survival. It was therefore important to better understand the causes of this downregulation through investigation of CD180 at the transcriptional and protein expression levels. Unexpectedly, we found CD180 RNA levels in CLL cells (n = 26) were comparable to those of normal B cells (n = 13), despite heterogeneously low expression of CD180 on the cell surface. We confirmed that CD180 RNA is translated into CD180 protein since cell surface CD180-negative cases presented with high levels of intracellular CD180 expression. Levels of MD-1 RNA were, however, significantly downregulated in CLL compared to normal controls. Together, these data suggest that changes in CD180 cell surface expression in CLL are not due to transcriptional downregulation, but defective post-translational stabilisation of the receptor due to MD-1 downregulation.

Evolution of LPS recognition and signaling: The bony fish perspective.

Fish are the most diverse and successful group of vertebrate animals, with about 30,000 species. The study of fish immunity is of great importance for understanding the evolution of vertebrate immunity, as they are the first animals to show both innate and adaptive immune responses. Although fish immunity is similar to that of mammals, there are obvious differences, such as their dependence of ambient temperature, their poor antibody response, and lack of antibody switching and lymph nodes. In addition, several important differences have also been found between the innate immune responses of fish and mammals. Among these, we will discuss in this review the high resistance of fish to the toxic effects of lipopolysaccharide (LPS) which can be explained by the absence of a Toll-like receptor 4 (Tlr4) ortholog in most fish species or by the inability of the Tlr4/Md2 (Myeloid differentiation 2) complex to recognize LPS, together with the presence of a negative regulator of the LPS signaling complex formed by the TLR-like molecule Rp105 (Radioprotective 105) and Md1. Taken together, these data support the idea that, although TLR4 and RP105 arose from a common ancestor to fish and tetrapods, the TLR4/MD2 receptor complex for LPS recognition arose after their divergence about 450 million years ago.

Cell surface expression of human RP105 depends on N-glycosylation of MD-1.

For its cell surface expression, radioprotective 105 (RP105) - an orphan Toll-like receptor - must form a complex with a soluble glycoprotein called myeloid differentiation 1 (MD-1). The number of RP105-negative cells is significantly increased in patients with systemic lupus erythematosus (SLE); however, to elucidate the mechanism underlying this increase, how RP105 is expressed on the cell surface depending on MD-1 should be investigated. We demonstrated that RP105 exhibits two forms depending on MD-1 and its two N-glycosylation sites, N96 and N156. Cell surface expression of RP105 decreased in the presence of mutant MD-1 (N96Q/N156Q). Nonglycosylated MD-1 decreased the de novo cell surface expression of RP105 but not pre-expressed RP105. Thus, the N-glycans of MD-1 may represent targets for SLE therapy.

LncRNA as a Therapeutic Target for Angiogenesis.

BACKGROUND: Out of 3 billion base pairs in human genome only ~2% code for proteins; and out of 180,000 transcripts in human cells, about 20,000 code for protein, remaining 160,000 are non-coding transcripts. Most of these transcripts are more than 200 base pairs and constitute a group of long non-coding RNA (lncRNA). Many of the lncRNA have its own promoter, and are well conserved in mammals. Accumulating evidence indicates that lncRNAs act as molecular switches in cellular differentiation, movement, apoptosis, and in the reprogramming of cell states by altering gene expression patterns. However, the role of this important group of molecules in angiogenesis is not well understood. Angiogenesis is a complex process and depends on precise regulation of gene expression. CONCLUSION: Dysregulation of transcription during this process may lead to several diseases including various cancers. As angiogenesis is an important process in cancer pathogenesis and treatment, lncRNA may be playing an important role in angiogenesis. In support of this, lncRNA microvascular invasion in hepatocellular carcinoma (MVIH) has been shown to activate angiogenesis. Furthermore, lncRNA-Meg3-knockout mouse showed increased expression of vascular endothelial growth factor pathway genes and increased cortical microvessel density. Overall, there is strong evidence that lncRNA is an important class of regulatory molecule, and a number of studies have demonstrated that these can be targeted to change cellular physiology and functions. In this review, we have attempted to summarize these studies and elucidate the potential of this novel regulatory molecule as a therapeutic target.

MD-1 deficiency attenuates dextran sodium sulfate (DSS)-induced colitis through modulating the function of colonic lamina propria dendritic cells.

Available evidence suggests that both dysregulated innate and adaptive immune pathways contribute to the aberrant intestinal inflammatory response in patients with inflammatory bowel disease (IBD). Myeloid Differentiation 1 (MD-1), also known as Lymphocyte Antigen 86 (Ly86), a secreted protein interacting with radioprotective 105 (RP105), plays an important role in Toll-like receptor 4 (TLR4) signaling pathway. Previous studies showed that MD-1 may be involved in the (patho) physiological regulation of the innate immune system and inflammation. In this study, we reported for the first time that MD-1 mRNA expression was up-regulated in both human IBD patients and DSS-treated WT mice. We showed that MD-1(-/-) mice were less susceptible to the development of colitis than WT controls as demonstrated by significantly reduced weight loss, disease activity index, colon histological scores, cellular infiltration and expression of inflammatory mediators. In addition, mucosal barrier function seemed to be intact in response to the loss of MD-1. Finally, lamina propria dendritic cells (LPDCs) from the colon of MD-1(-/-) mice after DSS exposure not only decreased in number but also significantly down-regulated the expression of surface maturation co-stimulatory molecules MHC-II, CD40 and CD86 compared with those from WT mice. Taken together, our results reveal that MD-1 deficiency is of critical importance in down-regulating induction and progression of DSS colitis, thereby suggesting that MD-1 might be a target for future interventional therapies of IBD.

The toll-like receptor family protein RP105/MD1 complex is involved in the immunoregulatory effect of exopolysaccharides from Lactobacillus plantarum N14.

The radioprotective 105 (RP105)/MD1 complex is a member of the Toll-like receptor (TLR) family. It was reported that RP105/MD1 cooperates with the lipopolysaccharide (LPS) receptor TLR4/MD2 complex and plays a crucial role in the response of immune cells to LPS. This work evaluated whether RP105, TLR4 or TLR2 were involved in the immunoregulatory capacities of Lactobacillus plantarum N14 (LP14) or its exopolysaccharides (EPS). EPS from LP14 were fractionated into neutral (NPS) and acidic (APS) EPS by anion exchange chromatography. Experiments with transfectant HEK(RP105/MD1) and HEK(TLR2) cells demonstrated that LP14 strongly activated NF-κB via RP105 and TLR2. When we studied the capacity of APS to activate NF-κB pathway in HEK(RP105/MD1) and HEK(TLR4) cells; we observed that APS strongly stimulated both transfectant cells. Our results also showed that LP14 and APS were able to decrease the production of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) in porcine intestinal epithelial (PIE) cells in response to enterotoxigenic Escherichia coli (ETEC) challenge. In order to confirm the role of TLR2, TLR4 and RP105 in the immunoregulatory effect of APS from LP14, we used small interfering RNA (siRNA) to knockdown these receptors in PIE cells. The capacity of LP14 and APS to modulate pro-inflammatory cytokine expression was significantly reduced in PIE(RP105-/-) cells. It was also shown that LP14 and APS were capable of upregulating negative regulators of the TLR signaling in PIE cells. This work describes for the first time that a Lactobacillus strain and its EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependend manner.

MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31.

Osteogenesis is the result of a complex sequence of events that involve the differentiation of mesenchymal stem cells (MSC) into osteoblasts. MSCs are multipotent adult stem cells that can give rise to different cell types of the mesenchymal germ layer. The differentiation fate of MSCs depends on the microenvironmental signals received by these cells and is tightly regulated by multiple pathways that lead to the activation of specific transcription factors. Among the transcription factors involved in osteogenic differentiation Osterix (Sp7) plays a key role and has been shown to be fundamental for bone homeostasis. However, the molecular events governing the expression of this transcription factor are not fully understood. In this study we set out to investigate the changes in the microRNA (miRNA) expression that occur during the osteogenic differentiation of bone marrow-derived MSCs. To this purpose, we analyzed the miRNA expression profile of MSCs deriving from 3 donors during the differentiation and mineralization processes by microarray. 29 miRNAs were significantly and consistently modulated during the osteogenic differentiation and 5 during the mineralization process. Interestingly, most of the differentially expressed miRNAs have been reported to be implicated in stemness maintenance, differentiation and/or oncogenesis. Subsequently, we focused our attention on the regulation of Osterix by miRNAs and demonstrated that one of the miRNAs differentially modulated during osteogenic differentiation, miR-31, controls Osterix expression through association to the 3' untranslated region of this transcription factor. By analyzing miR-31 and Osterix expression levels we found an inverse miRNA-target expression trend during osteogenic differentiation and in osteosarcoma cell lines. Moreover, the inhibition of the microRNA activity led to an increase in the endogenous expression of Osterix. Our results define a miRNA signature characterizing the osteogenic differentiation of MSCs and provide evidence for the involvement of miR-31 in the regulation of the bone-specific transcription factor Osterix.

Antagonistic functional duality of cancer genes.

Cancer evolution is a stochastic process both at the genome and gene levels. Most of tumors contain multiple genetic subclones, evolving in either succession or in parallel, either in a linear or branching manner, with heterogeneous genome and gene alterations, extensively rewired signaling networks, and addicted to multiple oncogenes easily switching with each other during cancer progression and medical intervention. Hundreds of discovered cancer genes are classified according to whether they function in a dominant (oncogenes) or recessive (tumor suppressor genes) manner in a cancer cell. However, there are many cancer "gene-chameleons", which behave distinctly in opposite way in the different experimental settings showing antagonistic duality. In contrast to the widely accepted view that mutant NADP(+)-dependent isocitrate dehydrogenases 1/2 (IDH1/2) and associated metabolite 2-hydroxyglutarate (R)-enantiomer are intrinsically "the drivers" of tumourigenesis, mutant IDH1/2 inhibited, promoted or had no effect on cell proliferation, growth and tumorigenicity in diverse experiments. Similar behavior was evidenced for dozens of cancer genes. Gene function is dependent on genetic network, which is defined by the genome context. The overall changes in karyotype can result in alterations of the role and function of the same genes and pathways. The diverse cell lines and tumor samples have been used in experiments for proving gene tumor promoting/suppressive activity. They all display heterogeneous individual karyotypes and disturbed signaling networks. Consequently, the effect and function of gene under investigation can be opposite and versatile in cells with different genomes that may explain antagonistic duality of cancer genes and the cell type- or the cellular genetic/context-dependent response to the same protein. Antagonistic duality of cancer genes might contribute to failure of chemotherapy. Instructive examples of unexpected activity of cancer genes and "paradoxical" effects of different anticancer drugs depending on the cellular genetic context/signaling network are discussed.