Osteopontin Summary

Osteopontin (OPN) is an extracellular matrix protein involved in inflammation. OPN is widely expressed and normally produced in bone, kidneys, and epithelial linings. Secreted OPN can be found in milk, urine, and blood. It has diverse functions in promoting cell migration, proliferation, survival, adhesion, and stimulating the infiltration of macrophages, neutrophils, dendritic cells, and T cells to inflammatory sites. OPN expression is increased during wound healing, fibrosis, and cancer and thus could serve as a useful biomarker for research in these areas.  More specifically, OPN has also gained popularity as a fibrosis marker in clinical research studies focused DILI, NASH and ALD as there is a lack of serological options to liver biopsy.  OPN is one a few translational liver markers able to be used in preclinical rodent models of a variety of liver diseases through relevant clinical research focused on acute and chronic liver disease.  Long story short, high OPN levels indicate more severe liver fibrosis.    Furthermore, OPN was named in a Letter of Support from FDA to the Predictive Safety Testing Consortium, as a biomarker to further explore for drug safety testing.

 

OPN Structure and general function

OPN is a secreted member of the SIBLING (small integrin-binding, ligand N-linked glycoprotein) family of glycoproteins.  It contains an Arg-Gly-Asp sequence (RGD domain) that binds members of the integrin transmembrane receptor family, including αvβ3 and αvβ5. In tumors and sites of injury or inflammation, OPN undergoes thrombin cleavage exposing a4 and a9 integrin binding sites (SVVYGLR in human and SLAYGLR in mouse/rat) on the N-terminal fragment (OPN N-Half).   As shown in Figure 1 below, the cleavage results in a C-terminal fragment (OPN C-Half) and the N-terminal fragment (OPN N-Half).  OPN C-Half interacts with the CD44 receptor on macrophages to induce chemotaxis.  Both full length OPN and OPN N-Half can bind to α4 integrin, however only OPN N-Half binds to α9 integrin.  Therefore, OPN N-Half functions include cell adhesion, migration, and collagen production, and are mediated through α4β1 and α9β1 integrin binding.  The OPN N-Half functions to accelerate the infiltration of immune cells, likely in response to the pro-inflammatory nature of thrombin activity.

 

OPN Protein

(also called FL-OPN or uncleaved OPN)

osteopontin
Figure 1   Osteopontin (OPN) is an extra cellular matrix protein involved with inflammation and flibrogenesis. It is a promising biomarker for many diseases including liver, kidney, oncology, etc. The protein is cleaved by thrombin to produce two products. The N-terminal product, N-Half, contains several integrin binding sites and are proposed to function in cell adhesion, migration and collagen production. The C-terminal product, C-Half, contains a CD44 binding site and acts to recruit macrophages and promote inflammation. Human, Mouse and Rat OPN ELISA products detect full length OPN, while N-Half OPN ELISA products are specific for the N-Half cleavage product.

 

Osteopontin & Liver Disease

In the healthy liver, OPN is expressed in the bile duct epithelium.  When the liver is exposed to acute or chronic damage induced by pathogens, toxicity or metabolites, signaling pathways that are typically dormant in healthy liver tissue becomes activated.  These pathways include TGFβ- 1, Hedgehog (Hh), and Wnt signaling pathways.  These pathways are critical for regeneration, but are also responsible for fibrosis and scarring.   Regulated by liver-repair signaling, OPN expression increases in hepatic stellate cells (HSCs), hepatic macrophages, hepatocytes, and necrotic liver tissues. As an inflammatory mediator, OPN functions as a chemoattractant for neutrophils and macrophages.  As a fibrosis mediator, OPN signals through integrin receptors on activated HSCs, increasing MAP kinase and NF‐κB signaling,resulting in the downstream increase of alpha‐smooth muscle actin (α‐SMA) and collagen 1a (COL1a) expression levels.  OPN induces higher levels of fibrosis markers, such as Collagen I and promotes increased HSC growth.  As shown in Figure 2, the repair signals collectively upregulate OPN to promote inflammation and fibrosis in the liver.

 

OPN in the Liver
osteopontin
Figure 2   Liver injuries triggered by acute and chronic damage activate dormant signals responsible for initiating liver repair. The TGFβ-1, Hedgehog (Hh), and Wnt signaling pathways are key and cooperative orchestrators of liver repair. Osteopontin (OPN) is extracellular matrix protein that is regulated by repair signals and a facilitator of inflammation and fibrosis. Reports show the OPN can be used as a potent biomaker for liver fibrosis and staging in pre-clinical and clinical research.

As a biomarker of fibrosis, and thus liver disease, OPN is an established serum marker.  Although it seems there are differential functions of full length vs N-Half OPN, there is no clear consensus if it is more relevant to measure full length vs N-Half, or vice versa.  Below are the research highlights of OPN as a biomarker in each liver disease field.

 

Drug-Induced-Liver-Injury

It is acknowledged that there is a need for more sensitive and mechanistically specific biomarkers to investigate drug safety during development.  Alanine aminotransferase (ALT) is a liver enzyme often measured to access liver function, but is not an optimal indicator of acute liver injury in that increases in serum ALT activity level are associated with other organ toxicities.  Thus, unidentified non-hepatic sources of serum ALT activity may inadvertently influence the results of drug safety testing.  As an alternative to measuring ALT, OPN has been named in an FDA Letter of Support for DILI biomarkers to assess the risk of progression of drug-induced liver injury (DILI) in subjects enrolled in clinical research trials.  Data suggests that OPN levels are able to predict subject death or need for liver transplantation and is a candidate early indicator to monitor liver damage.  Other markers that have been named in the FDA Letters of Support for the drug-induced liver injury safety biomarkers Cytokeratin 18 (CK-18), Total and hyperacetylated high mobility group protein B1 (HMGB1), and Macrophage colony-stimulating factor 1 receptor (CSF1R).  Liver Fatty Acid Binding Protein (L-FABP) is also an early marker of liver damage detected in the serum.

 

NASH (nonalcoholic steatohepatitis)

NASH is the more advanced form of non-alcoholic fatty liver disease (NAFLD). Elevated serum levels of OPN in NAFLD subjects are a significant predictor of portal inflammation.  In cases of human liver cirrhosis (LC), OPN N-Half levels are elevated compared to healthy subjects. OPN is significantly up-regulated and correlate with each stage of fibrosis in both human NASH and mouse models of NASH. Studies support that OPN is an important indicator and mediator of liver fibrosis in NASH.  Other common NASH biomarkers include ALT, AST, adiponectin, CRP, ProC3 and Cytokeratin 18 (CK-18).  Some novel and emerging NASH biomarkers are Noggin, Asporin, HA, Collagen IV, and FGF-21.

 

Alcoholic Liver Diseases (ALD)

Studies show that serum levels and gene expression levels of OPN are elevated and correlate with hepatic inflammation and fibrosis in human ALD.   Analyses of subjects with ALD also show strong correlation of OPN levels with neutrophil accumulation and expression of the fibrogenic factor TGF-β.  Due to the strong correlation between circulating OPN serum levels with hepatic OPN expression and fibrosis, OPN serves as a relevant biomarker for liver fibrosis in ALD.

 

Rat and Mouse OPN as a Translational Liver Biomarker

Similar to its role in humans, OPN functions as a chemoattractant for immune cells in animal models.  Overexpression of OPN in hepatocytes results in increased infiltration of mononuclear and CD8 positive cells into the liver.  Interestingly, transgenic overexpression of OPN in mouse hepatocytes drives the development of spontaneous liver fibrosis within 1 year.  Murine HSC-derived OPN interacts with αvβ3 integrin to activate the PI3K/pAkt/NF-κB signaling cascade which up-regulates collagen-I, ultimately driving liver fibrogenesis.  This suggests that in liver fibrogenesis, OPN promotes disease progression through integrin binding, downstream signaling pathway activation, and the eventual up-regulation of fibrotic markers, such as COL1a.  A similar mechanism of OPN activity has been determined in liver fibrogenesis rat models.  Given this mechanism of activity, OPN is a useful biomarker to consider for liver cirrhosis and fibrosis studies in preclinical models.

 

Rodent Models of Drug-Induced-Liver-Injury

In a DILI mouse model, OPN plays two roles during acetaminophen hepatotoxicity:  Intracellular OPN exhibits protective functions in hepatocytes by inhibiting acetaminophen metabolism.  However, after acetaminophen metabolism, secreted OPN promotes inflammation-induced liver damage through macrophage and neutrophil recruitment.  Following exposure to the hepatotoxic drug diethylnitrosamine (DEN), OPN-deficient mice are more susceptible to liver toxicity and estrogen-mediated hepatoprotection against DEN is impaired.  This finding suggests that during DEN-induced liver injury, OPN promotes hepatocyte survival and inhibits DEN metabolism.

As a biomarker for DILI in rodent models, OPN is an early indicator of acute liver damage.  The expression of OPN increased in Kupffer cells, hepatic macrophages, and stellate cells in rat liver following response to CCl4 treatment.   This increase in OPN expression is replicated in other acute liver injury models in rats, like Propionibacterium acnes treatment.  In both examples, mRNA increases peaked between 1 to 3 days.

 

Rodent Models of NASH

Animal models are being used to study the complex mechanisms driving NAFLD and NASH. In mouse models of methionine-choline deficient (MCD) diet-induced NASH fibrosis, OPN functions downstream of Wnt signaling by modulating TGF-β signaling to augment liver progenitor cell response and liver fibrosis.  However, in a murine obesity model of high fat-diet induced NAFLD, OPN mediates liver steatosis, inflammation, and macrophage accumulation. In fact, neutralizing OPN activity has been suggested as a therapeutic target to decrease obesity-associated liver inflammation and protect against hepatic steatosis during obesity-driven NAFLD.  Hepatic OPN expression is increased and drives fibrogenesis in mouse hepatic fibrosis models of bile duct ligations and MCD diets.  In efforts to overcome OPN-driven liver pathogenesis in mice, neutralizing OPN activity reduces the severity of liver fibrogenesis.

Circulating serum levels of OPN are being used as a biomarker in mouse models of NASH.  In a mouse model of fast food diet (FFD)-induced NAFLD, mice develop NASH with increasing fibrosis.  After 14 months, many of these mice develop primary liver cancer hepatocellular carcinoma (HCC).  In this study, increases in serum OPN were highly correlated with the incidence of HCC.   Another study analyzed MCD-induced NASH-fibrosis in mice resulting in the expected increases of Wnt pathway signaling activity and OPN expression.  Plasma levels of OPN reflect hepatic OPN expression and correlate with the degree of fibrosis in this animal NASH model, suggesting OPN as a useful biomarker of NASH-fibrosis.

 

Rodent Models of Alcoholic Liver Diseases (ALD)

In rat models of Alcoholic Steatohepatitis (ASH), OPN is expressed, interacts with α4β1 and α9β1 integrins, and contributes to hepatic neutrophil migration and liver injury.  In mice, OPN deficiency protects from alcohol-induced liver injury.  However, during alcoholic hepatitis (AH), OPN appears to function in protective roles rather than causal roles.  Ultimately further studies need to be done to clarify the function and mechanisms of OPN activity in alcoholic liver diseases.

In a rat model of ASH, increases of OPN protein and Opn mRNA correlate with degree of liver injury and neutrophil infiltration.  The increased Opn mRNA expression precedes neutrophil infiltration, suggesting potential as a predictive ASH biomarker. Studies using rat ASH models determined that elevated OPN levels drive neutrophil infiltration through the OPN N-half integrin binding motif, leading to eventual liver injury.  Due to the elevated levels of OPN N-Half in ASH models, together with OPN N-Half being a more potent chemoattractant than OPN, the measurement of OPN N-Half compared to OPN has value in predicting inflammatory liver diseases, including ASH.  More investigation is needed to correlate serum levels of OPN to the progression of liver disease in rodent ALD models.

 

Kit Summaries

Using the full length Osteopontin ELISA kit along with Osteopontin N-Half ELISA kit will enable the determination of the ratio of full length OPN to cleaved OPN N-Half.  This will provide insight into the mechanism of OPN activity.

  • Osteopontin (Human) ELISA #DPG27158
    • Measures full length OPN using the O-17 antibody as the capture antibody and 10A16 antibody as the detection antibody.
    • Solid phase sandwich ELISA
    • Application: EDTA-plasma, urine, or cell culture media.
    • For research use only.
  • Osteopontin N-Half ELISA (Human) #DPG27258
    • Measures the N-terminal fragment of OPN after thrombin cleavage using the 34E3 antibody as the capture antibody and the O-17 antibody as the detection antibody.
    • Solid phase sandwich ELISA
    • Application: EDTA-plasma, urine, synovial fluid, or cell culture media.
    • For research use only.
  • Osteopontin (Mouse) ELISA #DPG27351
    • Measures full length OPN using the O-17 antibody as the capture antibody and 0-165 antibody as the detection antibody.
    • Solid phase sandwich ELISA
    • Application: EDTA-plasma, urine, or cell culture media.
    • For research use only.
  • Osteopontin N-Half (Mouse) ELISA #DPG27259
    • Measures the N-terminal fragment of OPN after thrombin cleavage using the 34E3 antibody as the capture antibody and the O-17 antibody as the detection antibody.
    • Solid phase sandwich ELISA
    • Application: EDTA-plasma, urine, or cell culture media.
    • For research use only.
  • Osteopontin (Rat) ELISA #DPG27360
    • Measures full length OPN using the O-17 antibody as the capture antibody and 0-165 antibody as the detection antibody.
    • Solid phase sandwich ELISA
    • Application: EDTA-plasma, urine, or cell culture media.
    • For research use only.