White matter injury but not germinal matrix hemorrhage induces elevated osteopontin expression in human preterm brains.

Osteopontin (OPN) is a matricellular protein that mediates various physiological functions and is implicated in neuroinflammation, myelination, and perinatal brain injury. However, its expression in association with brain injury in preterm infants is unexplored. Here we examined the expression of OPN in postmortem brains of preterm infants and explored how this expression is affected in brain injury. We analyzed brain sections from cases with white matter injury (WMI) and cases with germinal matrix hemorrhage (GMH) and compared them to control cases having no brain injury. WMI cases displayed moderate to severe tissue injury in the periventricular and deep white matter that was accompanied by an increase of microglia with amoeboid morphology. Apart from visible hemorrhage in the germinal matrix, GMH cases displayed diffuse white matter injury in the periventricular and deep white matter. In non-injured preterm brains, OPN was expressed at low levels in microglia, astrocytes, and oligodendrocytes. OPN expression was significantly increased in regions with white matter injury in both WMI cases and GMH cases. The main cellular source of OPN in white matter injury areas was amoeboid microglia, although a significant increase was also observed in astrocytes in WMI cases. OPN was not expressed in the germinal matrix of any case, regardless of whether there was hemorrhage. In conclusion, preterm brain injury induces elevated OPN expression in microglia and astrocytes, and this increase is found in sites closely related to injury in the white matter regions but not with the hemorrhage site in the germinal matrix. Thus, it appears that OPN takes part in the inflammatory process in white matter injury in preterm infants, and these findings facilitate our understanding of OPN's role under both physiological and pathological conditions in the human brain that may lead to greater elucidation of disease mechanisms and potentially better treatment strategies.

WDR5-H3K4me3 epigenetic axis regulates OPN expression to compensate PD-L1 function to promote pancreatic cancer immune escape.

BACKGROUND: Despite PD-L1 (Programmed death receptor ligand-1) expression on tumor cells and cytotoxic T lymphocytes tumor infiltration in the tumor microenvironment, human pancreatic cancer stands out as one of the human cancers that does not respond to immune checkpoint inhibitor (ICI) immunotherapy. Epigenome dysregulation has emerged as a major mechanism in T cell exhaustion and non-response to ICI immunotherapy, we, therefore, aimed at testing the hypothesis that an epigenetic mechanism compensates PD-L1 function to render pancreatic cancer non-response to ICI immunotherapy. METHODS: Two orthotopic pancreatic tumor mouse models were used for chromatin immunoprecipitation-Seq and RNA-Seq to identify genome-wide dysregulation of H3K4me3 and gene expression. Human pancreatic tumor and serum were analyzed for osteopontin (OPN) protein level and for correlation with patient prognosis. OPN and PD-L1 cellular location were determined in the tumors using flow cytometry. The function of WDR5-H3K4me3 axis in OPN expression were determined by Western blotting. The function of H3K4me3-OPN axis in pancreatic cancer immune escape and response to ICI immunotherapy was determined in an orthotopic pancreatic tumor mouse model. RESULTS: Mouse pancreatic tumors have a genome-wide increase in H3K4me3 deposition as compared with normal pancreas. OPN and its receptor CD44 were identified being upregulated in pancreatic tumors by their promoter H3K4me3 deposition. OPN protein is increased in both tumor cells and tumor-infiltrating immune cells in human pancreatic carcinoma and is inversely correlated with pancreatic cancer patient survival. OPN is primarily expressed in tumor cells and monocytic myeloid-derived suppressor cells (M-MDSCs), whereas PD-L1 is expressed in tumor cells, M-MDSCs, polymorphonuclear MDSCs and tumor-associated macrophages. WDR5 is essential for H3K4me3-specific histone methyltransferase activity that regulates OPN expression in tumor cells and MDSCs. Inhibition of WDR5 significantly decreased OPN protein level. Inhibition of WDR5 or knocking out of OPN suppressed orthotopic mouse pancreatic tumor growth. Inhibition of WDR5 also significantly increased efficacy of anti-PD-1 immunotherapy in suppression of mouse pancreatic tumor growth in vivo. CONCLUSIONS: OPN compensates PD-L1 function to promote pancreatic cancer immune escape. Pharmacological inhibition of the WDR5-H3K4me3 epigenetic axis is effective in suppressing pancreatic tumor immune escape and in improving efficacy of anti-PD-1 immunotherapy in pancreatic cancer.

Recombinant Bovine and Human Osteopontin Generated by Chlamydomonas reinhardtii Exhibit Bioactivities Similar to Bovine Milk Osteopontin When Assessed in Mouse Pups Fed Osteopontin-Deficient Milk.

SCOPE: Osteopontin (OPN), a highly phosphorylated and glycosylated protein, is present in most body fluids, including milk. OPN appears at a high concentration in human milk (130-180 mg L-1 ), but not bovine milk (≈18 mg mL-1 ). It is previously shown that milk OPN is involved in various biological processes and therefore may be a valuable infant formula additive. METHODS AND RESULTS: In the present study, recombinant bovine OPN (rbOPN) and recombinant human OPN (rhOPN) are generated in a Chlamydomonas reinhardtii (C. reinhardtii) algal expression system. The rbOPN and rhOPN are phosphorylated but not glycosylated. To assess the bioactivities of rbOPN and rhOPN and compare their bioactivities to those of bovine milk OPN (bmOPN), wild-type (WT) mouse pups nursed by OPN knock-out (KO) dams are orally fed bmOPN, rbOPN, and rhOPN daily from postnatal days 1-21 (P1-21). Effects of these OPNs on development of the brain, intestine, and immune function are evaluated. The results show that rbOPN and rhOPN exhibit effects similar to those of bmOPN as well as mouse milk OPN on stimulating proliferation of the small intestine, increasing brain myelination and cognitive development, and enhancing development of immune function. CONCLUSION: rbOPN and rhOPN are likely to provide beneficial bioactivities when added to infant diets.

Tramadol aggravates cardiovascular toxicity in a rat model of alcoholism: Involvement of intermediate microfilament proteins and immune-expressed osteopontin.

Tramadol and alcohol are among commonly abused drugs. Although there are potential dangers reported upon their mixing, there are no previous reports describing this mixture's effects on the cardiovascular system (CVS). The aim was to study the effects of mixed alcohol and tramadol on the CVS of adult male rats. Fifty rats were divided into four groups: control, tramadol-treated group, alcohol-treated, and coadministration groups. Tramadol caused a significant increases in creatine kinase-MB, troponin I, malondialdehyde, protein carbonyl, 8-hydroxy-2'-deoxyguanosine, and a significant decrease in total antioxidant capacity with histological alterations in sections of the heart and aorta and a significant increase in the area% of collagen fibers while there was a nonsignificant difference in body weight, heart weight, heart weight/body weight ratio, lipid profile, tissue tumor necrosis factor-α and interferon-γ, intermediate microfilament proteins (IFPs) {desmin, vimentin, connexin43} gene expression, mean area% of elastic fibers in aortic tissue and osteopontin expression in cardiac and aortic tissue. Alcohol treatment caused a significant change in all the measured parameters and more damage in histological sections. The changes were highest in the coadministration group. There was a strong positive correlation between the area% of collagen fibers and vimentin gene expression, and the area% of osteopontin expression was positively correlated to connexin43 in cardiac and vascular tissue. Tramadol causes CVS injury mainly through oxidative stresses, while the alcohol effect is multifactorial; mixing both aggravates CVS injury. The study also highlights the role of IFPs and osteopontin-expression in inducing injury.

Influence of nano-hydroxyapatite coating implants on gene expression of osteogenic markers and micro-CT parameters. An in vivo study in diabetic rats.

This study evaluated the response of a nano-hydroxyapatite coating implant through gene expression analysis (runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), osteopontin (Opn), osteocalcin (Oc), receptor activator of nuclear factor-kappa B (Rank), receptor activator of nuclear factor-kappa B ligand (Rank-L), and osteoprotegerin (Opg)). Three-dimensional evaluation (percent bone volume (BV/TV); percent intersection surface (BIC); bone surface/volume ratio (BS/BV); and total porosity (To.Po)) were also analyzed. Mini implants were surgically placed in tibias of both healthy and diabetic rats. The animals were euthanized at 7 and 30 days. Evaluating all factors the relative expression of Rank showed that NANO surface presented the best results at 7 days (diabetic rats). Furthermore the levels of Runx2, Alp, Oc, and Opn suggest an increase in osteoblasts proliferation, especially in early stages of osseointegration. %BIC in healthy and diabetic (7 days) depicted statistically significant differences for NANO group. BV/TV, BS/BV and To.Po demonstrated higher values for NANO group in all evaluated time point and irrespective of systemic condition, but BS/BV 30 days (healthy rat) and 7 and 30 days (diabetic rat). Microtomographic and gene expression analyses have shown the benefits of nano-hydroxyapatite coated implants in promoting new bone formation in diabetic rats.

MAPK pathway and SIRT1 are involved in the down-regulation of secreted osteopontin expression by genistein in metastatic cancer cells.

AIM: The regulation of secreted osteopontin (OPN) expression by genistein and its functional sequel in the metastatic cancer cells (MDA-MB-435 and MDA-MB-231) was ascertained. MAIN METHODS: Western blot and Real-Time PCR were used to analyse the proteins and mRNA transcripts, respectively. Possible transcriptional regulation of secreted OPN was analyzed by chromatin immunoprecipitation assay, bioinformatics analysis, transfection and luciferase reporter assay. The specific siRNAs and constitutive p-ERKs were used to evaluate the role of the MAPK pathway. The functional sequel of genistein in these cells was analyzed by colony formation-, migration- and invasion- assay. KEY FINDINGS: Secreted OPN expression was inhibited (up to ~0.7-fold) by genistein in these cells. Genistein (50 µM) displayed a reduction in the aggressiveness of these cells concerning colony formation rate, migration, and invasion. The p-ERK½ was increased by ~2.5-fold and ~1.5-fold upon 50 µM genistein and 15 µM resveratrol treatments at 24 h, respectively. Knockdown of ERK½ and PD98059, the inhibitor of MEK, promoted secreted OPN expression in vitro in these cells; while, the transfection of the constitutive active ERK2 (L73P and S151D) decreased the secreted OPN expression. Further, silent mating type information regulation 2 homolog 1 (SIRT1) expression in the cells was increased (~1.6-fold) upon genistein treatment (50 µM) likewise with resveratrol (~1.5-fold), an activator for SIRT1. Knockdown of SIRT1 increased OPN mRNA transcripts expression level and secreted OPN protein level in these cells. SIGNIFICANCE: MAPK pathway and SIRT1 activation are involved in the regulation of secreted OPN by genistein in these cells.

Osteopontin-4 and Osteopontin-5 splice variants are expressed in several tumor cell lines.

Among osteopontin splice variants (OPN-SV), the expression profile of osteopontin-4 (OPN4) and osteopontin-5 (OPN5) has not been addressed in distinct cancer types. We herein aimed to investigate their expression in several cancer cell lines, besides comparing it in relation to the three previously described OPN-SV: OPNa, OPNb and OPNc. Total RNA from cancer cell lines, including prostate (PC3 and DU145), ovarian (A2780), breast (MCF-7 and MDA-MB-231), colorectal (Caco-2, HT-29 and HCT-116), thyroid (TT, TPC1 and 8505c) and lung (A549 and NCI-H460) was extracted, followed by cDNA synthesis. OPN-SV transcript analysis by RT-PCR or RT-qPCR were performed using OPN-SV specific oligonucleotides and gapdh and actin transcripts were used as housekeeping controls. OPN4 and OPN5 transcripts displayed co-expression in most tested cell lines. OPN4 was found expressed in similar or higher levels in relation to OPN5. Moreover, in most tested cell lines, OPN4 is also expressed in similar levels to OPNa or OPNb. The expression of OPN5 is also generally variable in relation to the other OPN-SV, but expressed in similar or higher levels in relation to OPNc, depending on each tested cell line. OPN4 and OPN5 seem to be co-expressed in several tumor types and OPN4 is one of the most overexpressed OPN-SV in distinct tumor cell lines. Once both OPN4 and OPN5 are differentially expressed and also evidence tumor-specific expression patterns, we hypothesize that similarly to the other OPN-SV, they also possibly contribute to key aspects of tumor progression, what should be further functionally investigated in distinct tumor models.

Osteopontin enhances the migration of lung fibroblasts via upregulation of interleukin-6 through the extracellular signal-regulated kinase (ERK) pathway.

Fibrosis is a phenomenon in which parenchyma is replaced with fibrous tissue. Persistent inflammation accompanied by dysregulation of cytokine production and repeated cycles of inflammation-associated tissue-repair induces fibrosis in various organs including the liver, lung, and kidney. In idiopathic pulmonary fibrosis, production of interleukin (IL)-6 and osteopontin (OPN) are dysregulated. Fibrosis leads to qualitative rather than quantitative changes of fibroblasts at the sites of tissue repair, and this leads to enlargement of fibrotic foci. These fibroblasts are immunohistochemically positive for OPN; however, the effect of overexpressed OPN in fibroblasts is not fully understood yet. In this study, we investigated the effect of OPN on IL-6 secretion and on migration and proliferation of fibroblasts. Lung fibroblasts overexpressing exogenous OPN showed that OPN was linked to the enhancement of cell migration through increased IL-6 secretion via the extracellular signal-regulated kinase (ERK) pathway. These results suggest that OPN may exert its pro-fibrotic functions, such as enhancement of fibroblasts migration by cooperating with chemoattractant IL-6, and may be involved in enlargement of fibrotic foci.

Periostin, tenascin, osteopontin isoforms in long- and non-long survival patients with pancreatic cancer: a pilot study.

Pancreatic adenocarcinoma (PDAC) is the most frequent histological type of malignancy in the pancreas. Extracellular matrix (ECM), plays a critical role during the process of human carcinogenesis and the possible diversity in matricellular proteins composition of ECM may have a significant impact on the clinical course of PDAC. Aim of this paper was to evaluate the expression of three matricellular proteins, including Periostin (POSTN), Tenascin (TNS) and Osteopontin (OPN), in PDAC from long-survival (LS) and non-long survival (NLS) patients. A total of 30 PDAC were analyzed, 15 from patients that survived more than 60 months after surgery (LS) and 15 that died from the disease within 24 (NLS). RNA was extracted and OPN, TNS and POSTN mRNA levels were evaluated by qRT-PCR. LS and NLS samples showed the same type of POSTN and TN isoforms. On the contrary, OPN seems to be preferentially expressed in NLS PDAC. Moreover, OPNb and OPNc isoforms were expressed exclusively in NLS samples. In conclusion, Our data led to hypothesize a possible relationship between the expression of different isoforms of each of these proteins and the clinical outcome of patients with PDAC.

Patterns of Osteopontin Expression in Abusive Head Trauma Compared with Other Causes of Pediatric Traumatic Brain Injury.

OBJECTIVE: To examine levels of plasma osteopontin (OPN), a recently described neuroinflammatory biomarker, in children with abusive head trauma (AHT) compared with children with other types of traumatic brain injury (TBI). STUDY DESIGN: The study cohort comprised children aged <4 years diagnosed with TBI and seen in the intensive care unit in a tertiary children's hospital. Patients were classified as having confirmed or suspected AHT or TBI by other mechanisms (eg, motor vehicle accidents), as identified by a Child Protection Team clinician. Serial blood samples were collected at admission and at 24, 48, and 72 hours after admission. Levels of OPN were compared across groups. RESULTS: Of 77 patients identified, 24 had confirmed AHT, 12 had suspected AHT, and 41 had TBI. There were no differences in the Glasgow Coma Scale score between the patients with confirmed AHT and those with suspected AHT and those with TBI (median score, 4.5 vs 4 and 7; P = .39). At admission to the emergency department, OPN levels were significantly higher in children with confirmed AHT compared with the other 2 groups (mean confirmed AHT, 471.5 ng/mL; median suspected AHT, 322.3 ng/mL; mean TBI, 278.0 ng/mL; P = .03). Furthermore, the adjusted mean trajectory levels of OPN were significantly higher in the confirmed AHT group compared with the other 2 groups across all subsequent time points (P = <.01). CONCLUSIONS: OPN is significantly elevated in children with confirmed AHT compared with those with suspected AHT and those with other types of TBI. OPN expression may help identify children with suspected AHT to aid resource stratification and triage of appropriate interventions for children who are potential victims of abuse.

Prostatic osteopontin expression is associated with symptomatic benign prostatic hyperplasia.

BACKGROUND: Male lower urinary tract symptoms (LUTS) occur in more than half of men above 50 years of age. LUTS were traditionally attributed to benign prostatic hyperplasia (BPH) and therefore the clinical terminology often uses LUTS and BPH interchangeably. More recently, LUTS were also linked to fibrogenic and inflammatory processes. We tested whether osteopontin (OPN), a proinflammatory and profibrotic molecule, is increased in symptomatic BPH. We also tested whether prostate epithelial and stromal cells secrete OPN in response to proinflammatory stimuli and identified downstream targets of OPN in prostate stromal cells. METHODS: Immunohistochemistry was performed on prostate sections obtained from the transition zone of patients who underwent surgery (Holmium laser enucleation of the prostate) to relieve LUTS (surgical BPH, S-BPH) or patients who underwent radical prostatectomy to remove low-grade prostate cancer (incidental BPH, I-BPH). Images of stained tissue sections were captured with a Nuance Multispectral Imaging System and histoscore, as a measure of OPN staining intensity, was determined with inForm software. OPN protein abundance was determined by Western blot analysis. The ability of prostate cells to secrete osteopontin in response to IL-1ß and TGF-ß1 was determined in stromal (BHPrS-1) and epithelial (NHPrE-1 and BHPrE-1) cells by enzyme-linked immunosorbent assay. Quantitative polymerase chain reaction was used to measure gene expression changes in these cells in response to OPN. RESULTS: OPN immunostaining and protein levels were more abundant in S-BPH than I-BPH. Staining was distributed across all cell types with the highest levels in epithelial cells. Multiple OPN protein variants were identified in immortalized prostate stromal and epithelial cells. TGF-ß1 stimulated OPN secretion by NHPrE-1 cells and both IL-1ß and TGF-ß1 stimulated OPN secretion by BHPrS-1 cells. Interestingly, recombinant OPN increased the mRNA expression of CXCL1, CXCL2, CXCL8, PTGS2, and IL6 in BHPrS-1, but not in epithelial cell lines. CONCLUSIONS: OPN is more abundant in prostates of men with S-BPH compared to men with I-BPH. OPN secretion is stimulated by proinflammatory cytokines, and OPN acts directly on stromal cells to drive the synthesis of proinflammatory mRNAs. Pharmacological manipulation of prostatic OPN may have the potential to reduce LUTS by inhibiting both inflammatory and fibrotic pathways.

NK cells induce hepatic ER stress to promote insulin resistance in obesity through osteopontin production.

High-fat diet (HFD) induced hepatic endoplasmic reticulum (ER) stress drives insulin resistance (IR) and steatosis. NK cells in adipose tissue play an important role in the pathogenesis of IR in obesity. Whether NK cells in the liver can induce hepatic ER stress and thus promote IR in obesity is still unknown. We demonstrate that HFD-fed mice display elevated production of proinflammatory cytokine osteopontin (OPN) in hepatic NK cells, especially in CD49a+ DX5- tissue-resident NK (trNK) cells. Obesity-induced ER stress, IR, and steatosis in the liver are ameliorated by ablating NK cells with neutralizing antibody in HFD-fed mice. OPN treatment enhances the expression of ER stress markers, including p-PERK, p-eIF2, ATF4, and CHOP in both murine liver tissues and HL-7702, a human liver cell line. Pretreatment of HL-7702 cells with OPN promotes hyperactivation of JNK and subsequent decrease of tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), resulting in impaired insulin signaling, which can be reversed by inhibiting ER stress. Collectively, we demonstrate that hepatic NK cells induce obesity-induced hepatic ER stress, and IR through OPN production.

1,2,3-Triazole tethered 1,2,4-trioxanes: Studies on their synthesis and effect on osteopontin expression in MDA-MB-435 breast cancer cells.

Artemisinin and its analogs have shown potent anticancer activity in primary cancer cultures and cell lines by inhibiting cancer proliferation, metastasis, and angiogenesis. Despite its apparent compatibility to normal cells and low IC50 values in comparison to the commonly used anticancer drugs, the underlying mechanisms behind their cytotoxic effects are not yet fully understood. Surprisingly, the efficacy of synthetic 1,2,4-trioxanes against cancer has not been explored yet. Given the high antitumor activity of artemisinin dimers in comparison to their monomers, we report here the synthesis of simple 1,2,3-triazole conjugated 1,2,4-trioxanes and their potential antitumor activity by studying their inhibitory effect on osteopontin (OPN) expression in MDA-MB-435 breast cancer cells. It may be noted that despite being a strong marker to identify human tumor metastasis, no study on effect of artemisinin and its synthetic and semisynthetic derivatives on OPN expression has ever been studied. Although our initial studies did not notice any straight-line relationship between the number of trioxane units in a molecule to the extent of inhibition of OPN protein expression, we could observe better results in some cases in comparison to artemisinin. We have observed that artemisinin did not show appreciable OPN downregulation in MDA-MB-435 cancer cells, but dihydroartemisinin (DHA) and some synthetic 1,2,4-trioxane monomers and dimers showed downregulation of OPN expression. Therefore, these compounds may act as an anti-metastatic agent in controlling breast cancer cells metastasis.

Association between the expression of secreted phosphoprotein - related genes and prognosis of human cancer.

BACKGROUND: While many studies have assessed the predictive value of secreted phosphoprotein (SPP) genes in cancer, the findings have been inconsistent. To resolve these inconsistencies, we systematically analyzed the available data to determine whether SPP1 and SPP2 are prognostic markers in the context of human cancer. METHODS: The expression of SPP1 and SPP2 was assessed by Oncomine analysis. The PrognoScan database was used to assess the prognostic value of SPP1 and SPP2, with cBioPortal used to assess copy number variations. The STRING database was used to generate a Protein - Protein Interaction (PPI) network for SPP genes. RESULTS: SPP1 was more likely to be over-expressed in breast, bladder, colorectal, head, neck, liver, lung, and esophageal cancers. SPP2 was expressed at lower levels in colorectal cancer, leukemia, liver cancer and pancreatic cancer. In addition, SPP1 and SPP2 mutations mainly occurred in cutaneous melanoma and endometrial cancer. CONCLUSIONS: Our results suggest that SPP1 and SPP2 may be effective therapeutic or diagnostic targets in certain cancers. Further research is required to confirm these results and verify the value of SPP1 and SPP2 as clinical markers of cancer prognosis.

Direct electrical stimulation enhances osteogenesis by inducing Bmp2 and Spp1 expressions from macrophages and preosteoblasts.

The capability of electrical stimulation (ES) in promoting bone regeneration has already been addressed in clinical studies. However, its mechanism is still being investigated and discussed. This study aims to investigate the responses of macrophages (J774A.1) and preosteoblasts (MC3T3-E1) to ES and the faradic by-products from ES. It is found that pH of the culture media was not significantly changed, whereas the average hydrogen peroxide concentration was increased by 3.6 and 5.4 µM after 1 and 2 hr of ES, respectively. The upregulation of Bmp2 and Spp1 messenger RNAs was observed after 3 days of stimulation, which is consistent among two cell types. It is also found that Spp1 expression of macrophages was partially enhanced by faradic by-products. Osteogenic differentiation of preosteoblasts was not observed during the early stage of ES as the level of Runx2 expression remains unchanged. However, cell proliferation was impaired by the excessive current density from the electrodes, and also faradic by-products in the case of macrophages. This study shows that macrophages could respond to ES and potentially contribute to the bone formation alongside preosteoblasts. The upregulation of Bmp2 and Spp1 expressions induced by ES could be one of the mechanisms behind the electrically stimulated osteogenesis.

Osteopontin attenuates early brain injury through regulating autophagy-apoptosis interaction after subarachnoid hemorrhage in rats.

AIM: To determine the effect of osteopontin (OPN) on autophagy and autophagy-apoptosis interactions after SAH. METHODS: The endovascular perforation model of SAH or sham surgery was performed in a total of 86 Sprague-Dawley male rats. The temporal expressions of endogenous OPN and autophagy-related proteins (Beclin 1, ATG5, LC3 II to I ratio) were measured in sham and SAH rats at different time points (3, 6, 12, 24, and 72 hours). Rats were randomly divided into three groups: Sham, SAH + Vehicle (PBS, phosphate-buffered saline), and SAH + rOPN (5 µg/rat recombinant OPN). Neurobehavioral tests were performed 24 hours after SAH, followed by the collection of brain samples for assessment of autophagy and apoptosis proteins. These tests assessed whether an autophagy-apoptosis relationship existed on the histological level in the brain. RESULTS: Endogenous OPN and autophagy-related proteins all increased after SAH. rOPN administration improved neurological dysfunction, increased the expression of autophagy-related proteins (Beclin 1, ATG5, LC3 II to I ratio) and antiapoptotic protein Bcl-2, while decreasing the expression of proapoptotic proteins (cleaved Caspase-3 and Bax). rOPN also regulated autophagy-apoptosis interactions 24 hours after SAH. CONCLUSION: rOPN attenuates early brain injury and inhibits neuronal apoptosis by activating autophagy and regulating autophagy-apoptosis interactions.

Kidney Injury Molecule-1 Is Upregulated in Renal Lipotoxicity and Mediates Palmitate-Induced Tubular Cell Injury and Inflammatory Response.

Diabetic nephropathy is increasingly recognized as a major contributor to kidney failure in patients with obesity and type 2 diabetes. This study was designed to identify the molecular mediators of kidney injury associated with metabolic syndrome with or without hyperglycemia. We compared renal gene expression profiles in Zucker lean (ZL), Zucker obese (ZO), and Zucker diabetic (ZD) rats using cDNA microarray with quantitative verification of selected transcripts by real-time PCR. Compared to the 20-week-old ZL control (glucose: 110 ± 8 mg/dL), both prediabetic ZO (glucose: 157 ± 11 mg/dL) and diabetic ZD (glucose: 481 ± 37 mg/dL) rats displayed hyperlipidemia and kidney injury with a high degree of proteinuria. cDNA microarray identified 25 inflammation and injury-related transcriptomes whose expression levels were similarly increased in ZO and ZD kidneys. Among them, kidney injury molecule-1 (KIM-1) was found to be the most highly upregulated in both ZO and ZD kidneys. Immunofluorescence staining of kidney sections revealed a strong correlation between lipid overload and KIM-1 upregulation in proximal tubules of ZO and ZD rats. In cultured primary renal tubular epithelial cells (TECs), administration of saturated fatty acid palmitate resulted in an upregulation of KIM-1, osteopontin, and CD44, which was greatly attenuated by U0126, an inhibitor of extracellular signal-regulated kinase (ERK)1/2. Moreover, knockdown of KIM-1 by siRNA interference inhibited palmitate-induced cleaved caspase-3, osteopontin, and CD44 proteins in primary TECs. Our results indicate that KIM-1 expression is upregulated in renal lipotoxicity and may play an important role in fatty acid-induced inflammation and tubular cell damage in obesity and diabetic kidney disease.

Low-dose cadmium exposure acts on rat mesenchymal stem cells via RANKL/OPG and downregulate osteogenic differentiation genes.

Chronic cadmium (Cd) toxicity is a significant health concern, and the mechanism of long-term low-dose Cd exposure on bone has not been fully elucidated till date. This study aimed to assess the association between rat mesenchymal stem cells (MSCs) and long-term Cd exposure through 38-week intake of CdCl2 at 1 and 2 mg/kg body weight (bw). Increased gene expression of receptor activator of NF-κB ligand (RANKL) and decreased gene expression of osteoprotegerin (OPG) were observed. Fold change of RANKL gene expression (fold change = 1.97) and OPG gene expression (fold change = 1.72) showed statistically significant differences at dose 2 mg/kg bw. Decreased expression of key genes was observed during the early osteogenic differentiation of MSCs. The gene expression of Osterix in 1 mg/kg bw group was decreased by 3.70-fold, and the gene expressions of Osterix, Osteopontin, collagen type I alpha 2 chain (COL1a2) and runt-related transcription factor 2 (RUNX2) in 2 mg/kg bw group were decreased by 1.79, 1.67, 1.45 and 1.35-folds, respectively. Exposure to CdCl2 induced an increase in the renal Cd load, but only an adaptive response was observed, including increased expression of autophagy-related proteins LC3B and Beclin-1, autophagy receptor p62, and heme oxygenase 1 (HO-1), which is an inducible isoform that releases in response to stress. There were no significant changes in the urinary low molecular weight proteins including N-acetyl-b-D-glucosaminidase (NAG), ß2-microglobulin and albumin (U-Alb). Urinary calcium (Ca) excretion showed no increase, and no obvious renal histological changes. Taken together, these results indicated that the chronic CdCl2 exposure directly act on MSCs through RANKL/OPG pathway and downregulate the key genes involved in osteogenic differentiation of MSCs. The toxic effect of Cd on bone may occur in parallel to nephrotoxicity.

Osteopontin plays important roles in pulmonary arterial hypertension induced by systemic-to-pulmonary shunt.

Recent studies indicated that osteopontin (OPN) was involved in the genesis and progression of pulmonary arterial hypertension (PAH); however, its role in congenital heart disease-associated PAH (CHD/PAH) remains unknown. Our results showed that OPN was increased in lungs and plasma of patients with Eisenmenger syndrome; moreover, OPN and αVß3-integrin expression levels were augmented in rat lungs exposed to systemic-to-pulmonary shunt. Cell culture assay demonstrated that distal pulmonary arterial smooth muscle cells (PASMCs) from rat lungs suffering from volume and pressure overload exhibited enhanced proliferation compared with those from healthy rats. Mechanical stretch (20% at 1 Hz) increased OPN expression and activated ERK1/2 and protein kinase B (Akt) signal pathway in distal PASMCs from healthy rats. Interestingly, OPN enhanced the proliferation and migration of PASMCs while blocking αVß3-integrin with neutralizing antibody LM609 or Arg-Gly-Asp peptidomimetic antagonist cyclo(Ala-Arg-Gly-Asp-3-aminomethylbenzoyl) (XJ735), rectified the proliferative and migratory effects of OPN, which were partially mediated via ERK1/2 and Akt signaling pathways. Furthermore, surgical correction of systemic-to-pulmonary shunt, particularly XJ735 supplementation after surgical correction of systemic-to-pulmonary shunt, significantly alleviated the pulmonary hypertensive status in terms of pulmonary hemodynamic indices, pulmonary vasculopathy, and right ventricular hypertrophy. In summary, OPN alteration in lungs exposed to systemic-to-pulmonary shunt exerts a deteriorative role in pulmonary vascular remodeling through modulating the proliferation and migration of PASMCs, at least in part, via ανß3-ERK1/2 and ανß3-Akt signaling pathways. Antagonizing OPN receptor ανß3-integrin accelerated the regression of pulmonary vasculopathy after surgical correction of systemic-to-pulmonary shunt, indicating a potential therapeutic strategy for patients with CHD/PAH.-Meng, L., Liu, X., Teng, X., Gu, H., Yuan, W., Meng, J., Li, J., Zheng, Z., Wei, Y., Hu, S. Osteopontin plays important roles in pulmonary arterial hypertension induced by systemic-to-pulmonary shunt.

JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).