Ectonucleotide Pyrophosphatase/phosphodiesterase 1 Expression Research Articles

Abstract CT161: First-in-human study of ENPP1 inhibitor, SR-8541A, for the treatment of solid tumors

Abstract Purpose: Compelling evidence suggests that careful and therapeutically relevant activation of the STING (STimulator of INterferon Genes) pathway is vital to elicit potent anti-cancer innate immune responses. STING is widely expressed and targeting it directly poses many challenges, including systemic activation of interferon. Therefore, alternative approaches to activate STING in a controlled manner may generate a better therapeutic response in cancer patients. Ectonucleotide Pyrophosphatase/Phosphodiesterase-1 (ENPP1) is the direct negative regulator of the STING pathway that hydrolyzes 2’3’ cGAMP, the direct activator of STING. Highest levels of 2′3′ cGAMP can be found in tumors and recent evidence suggests that 2′3′ cGAMP acts locally, as a paracrine immune transmitter. Recent studies have also reported elevated ENPP1 expression in metastatic tumors. We hypothesize that inhibiting ENPP1 with a small molecule inhibitor may produce superior outcomes by activating STING in the tumor microenvironment. We have developed SR-8541A, a highly selective and potent inhibitor of ENPP1. Our initial work to support the development of SR-8541A consisted of pharmacodynamics studies, bioanalytical development, pharmacokinetics (PK) and non-GLP toxicology studies in three species, a GLP repeat dose dog telemetry study, and 28-day GLP toxicology studies in rats and dogs including toxicokinetic (TK) analyses. Here we report initial findings from our ongoing first in human, phase I trial of SR-8541A in advanced metastatic solid tumors. Methods: This study is evaluating the safety, tolerability, and pharmacokinetics (PK) of SR-8541A administered orally as a monotherapy in subjects with solid tumors which are refractory to standard therapeutic options, or for which there are no standard therapeutic options (NCT06063681). The primary objective of the study is to characterize the safety, tolerability, DLTs, maximum tolerated dose and RP2D and schedule for future studies of SR-8541A. Secondarily, the study aims to evaluate the PK and efficacy per RECIST of SR-8541A. SR-8541A is administered orally in 28-day cycles and is following an accelerated titration dose (ATD) escalation. Blood samples are being collected for PK assessment, target engagement, and biomarker assessment. Results: Sample analysis from a subject treated with SR-8541A (5 mg) in the ATD portion of the study is ongoing. No treatment-related adverse events have been reported. No dose-limiting toxicities occurred during the 28-day DLT period. Plasma drug levels collected for the PK show cMAX concentrations in the projected therapeutic dose range. Biomarker assessment is ongoing and will be included in the presentation. The SR-8541A dose has escalated and the study is currently enrolling at the 10 mg dose. Conclusions: The first evaluated dose of SR-8541A has shown to be safe and well tolerated. Pharmacokinetic data shows strong oral bioavailability and supports escalation in the phase I trial and continued assessment of SR-8541A in the clinic. Citation Format: Alexis S. Weston, Trason Thode, Monil Shah, Amanda Seiz, Jonathan Northrup, Srinivas Kasibhatla, Mohan Kaadige, Sunil Sharma. First-in-human study of ENPP1 inhibitor, SR-8541A, for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT161.

ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

Abstract 1330: Role of ENPP1 mediated extracellular cGAMP hydrolysis in cancer metastasis and immune evasion

Abstract Chromosome instability (CIN) promotes metastasis through sustained tumor-cell autonomous response to cytosolic DNA. Chromosome mis-segregation engenders the formation of micronuclei, which upon rupture chronically activates the cGAS-STING DNA sensing pathway. Tumor cGAS recognizes cytosolic DNA to generate cGAMP, a potent immune-stimulatory molecule that is readily exported into the extracellular space to enforce a type 1 IFN-mediated anti-tumor response in a host STING dependent manner. However, it is unclear how highly aggressive, unstable tumors have evolved to co-opt this chronic inflammatory signaling to drive tumorigenic behaviors and immune evasion. Here, we show that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) may drive tumor metastasis through degradation of extracellular cGAMP. Using RNA sequencing of isogenic tumors, we uncovered a link between ENPP1 and CIN. We leveraged syngeneic mouse models to demonstrate that genetic perturbation of tumor ENPP1 and overexpression of a catalytically inactive ENPP1 mutant suppress metastasis. Furthermore, loss of ENPP1 increases tumor immune infiltration, reduces extracellular adenosine, and enhances response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. In line with these findings, ENPP1 expression correlates with immune suppression in human cancers. These results suggest that ENPP1-mediated hydrolysis of cGAMP may facilitate metastasis through evasion of immune surveillance, perhaps in part through cGAMP breakdown into immune-suppressive adenosine, in which the contributions of tumor and host adenosine can be further explored. Taken together, our study demonstrates how ENPP1-mediated hydrolysis of cGAMP can transform an otherwise inflammatory pathway into an immune-suppressive mechanism to promote tumor progression and metastasis. Citation Format: John Young Ho Kwon, Jun Li, Mercedes Duran, Ninjit Dhanota, Samuel Bakhoum. Role of ENPP1 mediated extracellular cGAMP hydrolysis in cancer metastasis and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1330.

Abstract 53: Discovery of novel potent and orally bioavailable small-molecule inhibitors of ENPP1 to stabilize cGAMP and ATP in the tumor microenvironment and boost anti-tumor immunity

Abstract Extracellular 2',3'-cyclic GMP-AMP (cGAMP) has a critical immune-transmitter role in the tumor microenvironment (TME). Tumor cells produce and secrete cGAMP, which primes immune cells for tumor rejection through STING (stimulator of interferon genes) signalling (Marcus et al., Immunity 2018; Carozza et al., Nature Cancer 2020). Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) is the only enzyme known to hydrolyze extracellular cGAMP. In concert with the ectoATPase CD39 and the 5'-nucleotidase CD73, ENPP1 also contributes to the generation of an immunosuppressive TME by converting extracellular ATP into adenosine. ENPP1 is overexpressed in a number of tumor types, including breast cancer, liver cancer, thyroid cancer and sarcomas, and has been associated with poor outcome. Hence, inhibition of ENPP1 is an emerging strategy to augment anti-tumor immunity by stabilizing extracellular cGAMP and ATP, thereby turning cold tumors into immunologically hot tumors. Here, we report on the identification and characterization of novel chemical series of ENPP1 inhibitors. A drug-like small-molecule library (~160,000 compounds) was screened to identify inhibitors of ENPP1-mediated cGAMP hydrolysis. Hits were confirmed to also inhibit ATP hydrolysis by ENPP1, but not nucleotide hydrolysis mediated by the closely related ENPP2 (autotaxin) enzyme. A selection of hits, chemically distinct from previously reported ENPP1 inhibitors (such as those based on QS1; Carroza et al, Cell Chem Biol, 2020), were subjected to hit-to-lead optimization supported by structure-based guidance. This led to the identification of ENPP1 inhibitors with sub-nM potency on ENPP1 in biochemical assays, which maintained a >1000x selectivity window with respect to ENPP2 and to other phosphodiesterases. These compounds translated well to stabilization of nucleotides in the presence of ENPP1-overexpressing cancer cell lines, with IC50 values in the nM range. A selection of compounds was profiled for DMPK (drug metabolism and pharmacokinetic) parameters, and compounds with a suitable profile were prioritized for in vivo evaluation. Orally bio-available, metabolically stable compounds were assessed in mouse syngeneic tumor models, selected on the basis of high ENPP1 and high cGAS (cGAMP synthase) expression. Stabilization of cGAMP, activation of a STING-mediated cytokine response and immune-cell infiltration/activation in the TME were used as pharmacodynamic endpoints. An update on in vivo efficacy data in a range of ENPP1-positive tumor models will be provided during the presentation. These novel orally bioavailable ENPP1 inhibitors unleash local, TME-restricted, innate immune activation, and hold the promise to overcome the current limitations of direct STING agonists. Citation Format: Matthias Versele, Javier del Pino Garcia, Ilse Vandecaetsbeek, Karolien Castermans, Ranie Kellens, Wanda Haeck, Hugo Klaassen, Arnaud Bourin, Dries De Clercq, Sara Allasia, Sandro Boland, Arnaud Marchand, Patrick Chaltin, Mathieu Bollen. Discovery of novel potent and orally bioavailable small-molecule inhibitors of ENPP1 to stabilize cGAMP and ATP in the tumor microenvironment and boost anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 53.

The role of ENPP1/PC-1 in osteoinduction by calcium phosphate ceramics

In the past decade, calcium phosphate (CaP) ceramics have emerged as alternatives to autologous bone grafts for the treatment of large, critical-sized bone defects. In order to be effective in the regeneration of such defects, ceramics must show osteoinductive behaviour, defined as the ability to induce de novo heterotopic bone formation. While a set of osteoinductive CaP ceramics has been developed, the exact processes underlying osteoinduction, and the role of the physical and chemical properties of the ceramics, remain largely unknown. Previous studies have focused on the role of the transcriptome to shed light on the mechanism of osteoinduction at the mRNA level. To complement these studies, a proteomic analysis was performed to study the behaviour of hMSCs on osteoinductive and non-osteoinductive CaPs. The results of this analysis suggest that plasma cell glycoprotein 1 (PC-1), encoded by the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene, plays a key role in the process of osteoinduction by CaP ceramics. Validation experiments have confirmed that indeed, the mRNA expression of ENPP1 and the production of PC-1 are higher on osteoinductive than on non-osteoinductive CaP ceramics, a trend that was also observed for other osteogenic markers such as bone morphogenetic protein 2 (BMP2) and osteopontin (OPN), but not for alkaline phosphatase (ALP). Our results also showed that the expression of PC-1 is restricted to those cells which are in direct contact with the CaP ceramic surface, plausibly due to the localised depletion of calcium and inorganic phosphate ions from the supersaturated cell culture medium as CaP crystallises on the ceramic surface. Replicating the surface of the osteoinductive ceramic in polystyrene resulted in a significant decrease in ENPP1 expression, suggesting that surface structural properties alone are not sufficient to induce ENPP1 expression. Finally, knocking down ENPP1 expression in hMSCs resulted in increased BMP2 expression, both at the mRNA and protein level, suggesting that ENPP1 is a negative regulator of BMP-2 signalling. Taken together, this study shows, for the first time, that ENPP1/PC-1 plays an important role in CaP-induced osteogenic differentiation of hMSCs and thus possibly osteoinduction by CaP ceramics. Furthermore, we have identified a crucial role for the interfacial (chemical) events occurring on the CaP ceramic surface in the process of osteoinduction. This knowledge can contribute to the development of new bone graft substitutes, with improved osteoinductive potential.

Hypercementosis Associated with ENPP1 Mutations and GACI

Mineralization of bones and teeth is tightly regulated by levels of extracellular inorganic phosphate (Pi) and pyrophosphate (PPi). Three regulators that control pericellular concentrations of Pi and PPi include tissue-nonspecific alkaline phosphatase (TNAP), progressive ankylosis protein (ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Inactivation of these factors results in mineralization disorders affecting teeth and their supporting structures. This study for the first time analyzed the effect of decreased PPi on dental development in individuals with generalized arterial calcification of infancy (GACI) due to loss-of-function mutations in the ENPP1 gene. Four of the 5 subjects reported a history of infraocclusion, overretained primary teeth, ankylosis, and/or slow orthodontic tooth movement, suggesting altered mineral metabolism contributing to disrupted tooth movement and exfoliation. All subjects had radiographic evidence of unusually protruding cervical root morphology in primary and/or secondary dentitions. High-resolution micro–computed tomography (micro-CT) analyses of extracted primary teeth from 3 GACI subjects revealed 4-fold increased cervical cementum thickness (P = 0.00007) and a 23% increase in cementum density (P = 0.009) compared to age-matched healthy control teeth. There were no differences in enamel and dentin densities between GACI and control teeth. Histology revealed dramatically expanded cervical cementum in GACI teeth, including cementocyte-like cells and unusual patterns of cementum resorption and repair. Micro-CT analysis of Enpp1 mutant mouse molars revealed 4-fold increased acellular cementum thickness (P = 0.002) and 5-fold increased cementum volume (P = 0.002), with no changes in enamel or dentin. Immunohistochemistry identified elevated ENPP1 expression in cementoblasts of human and mouse control teeth. Collectively, these findings reveal a novel dental phenotype in GACI and identify ENPP1 genetic mutations associated with hypercementosis. The sensitivity of cementum to reduced PPi levels in both human and mouse teeth establishes this as a well-conserved and fundamental biological process directing cementogenesis across species (ClinicalTrials.gov NCT00369421).

Abnormal Mechanical Loading Induces Cartilage Degeneration by Accelerating Meniscus Hypertrophy and Mineralization After ACL Injuries In Vivo

Background: Although patients with an anterior cruciate ligament (ACL) injury have a high risk of developing posttraumatic osteoarthritis (PTOA), the role of meniscus hypertrophy and mineralization in PTOA after an ACL injury remains unknown. Purpose/Hypothesis: The purpose of this study was to determine if menisci respond to abnormal loading and if an ACL injury results in meniscus hypertrophy and calcification. The hypotheses were that (1) abnormal mechanical loading after an ACL injury induces meniscus hypertrophy and mineralization, which correlates to articular cartilage damage in vivo, and (2) abnormal mechanical loading on bovine meniscus explants induces the overexpression of hypertrophic and mineralization markers in vitro. Study Design: Controlled laboratory study. Methods: In vivo guinea pig study (hypothesis 1): Three-month-old male Hartley guinea pigs (n = 9) underwent ACL transection (ACLT) on the right knee; the left knee served as the control. Calcification in the menisci was evaluated by calcein labeling 1 and 5 days before knee harvesting at 5.5 months. Cartilage and meniscus damage and mineralization were quantified by the Osteoarthritis Research Society International score and meniscus grade, respectively. Indian hedgehog (Ihh), matrix metalloproteinase–13 (MMP-13), collagen type X (Col X), progressive ankylosis homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase–1 (ENPP1), alkaline phosphatase (ALP), inorganic pyrophosphate (PPi), and inorganic phosphate (Pi) concentrations were evaluated by immunohistochemistry and enzyme-linked immunosorbent assay. In vitro bovine meniscus explant study (hypothesis 2): Bovine meniscus explants were subjected to 25% strain at 0.3 Hz for 1, 2, and 3 hours. Cell viability was determined using live/dead staining. The levels of mRNA expression and protein levels were measured using real-time quantitative reverse transcription polymerase chain reaction and Western blot after 24, 48, and 72 hours in culture. The conditioned medium was collected for sulfated glycosaminoglycan (GAG) release and Pi/PPi assay. Results: In vivo guinea pig study: Meniscus size and area as well as intensity of meniscus calcification were significantly increased in the ACLT group compared with the control group. Both calcified area and intensity were correlated with cartilage damage in the ACLT group (meniscus calcified area: r = 0.925, P < .0001; meniscus calcified intensity: r = 0.944, P < .0001). Ihh, MMP-13, Col X, ANKH, ENPP1, and ALP expression were increased in the ACLT group compared with the control group. The Pi level and Pi/PPi ratio increased by 63% and 42%, respectively, in the ACLT group compared with the control group. In vitro bovine meniscus explant study: Cell death was found in the superficial zone of the bovine meniscus explants after loading for 3 hours. The mRNA expression and protein levels of MMP-13, ANKH, ENPP1, and ALP were up-regulated in all 3-hour loaded samples. The Pi/PPi ratio and sulfated GAG content in the culture medium were increased in the 3-hour loaded group. Conclusion: Meniscus hypertrophy and mineralization correlated to cartilage degeneration after ACL injuries. Clinical Relevance: The study data suggest that the suppression of meniscus hypertrophy and calcification may decrease the risk of PTOA after ACL injuries.

Liver ENPP1 protein increases with remission of type 2 diabetes after gastric bypass surgery.

BackgroundType 2 diabetes mellitus (T2DM) is a progressive disease resulting from increasing insulin resistance and reduced pancreatic β-cell insulin secretion. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibits insulin signalling and may contribute to the pathogenesis of T2DM. Others have found elevated ENPP1 levels in muscle, fat, and skin tissues from insulin resistant individuals, but similar data on liver ENPP1 is lacking. The purpose of this study was to compare expression and protein concentrations of ENPP1 in liver between patients with and without T2DM.MethodsRoux-en-Y gastric bypass surgery (RYGB) results in remission of insulin resistance and T2DM thus presenting an opportunity to examine some critical aspects of these conditions. We measured liver ENPP1 gene and protein expression in individuals with or without T2DM at RYGB and on average 17 (±5.6) months later.ResultsWe found liver ENPP1 protein abundance was lower in individuals with T2DM than in those with normal glucose tolerance, and increased after RYGB surgery in those individuals who had remission of T2DM. ENPP1 positively correlated with insulin sensitivity at the liver (as measured by HOMA-IR), which is contrary to what others have reported in other insulin target tissues.ConclusionsLiver ENPP1 expression in T2DM is the reverse of that expected based on expression in other tissues and is likely due to the unique role the liver has in insulin clearance. The work presented here adds another dimension to the role of ENPP1, and supports the hypothesis that ENPP1 may act as a natural modulator of insulin signalling in the liver.Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-014-0222-x) contains supplementary material, which is available to authorized users.

Stem cell characteristics in glioblastoma are maintained by the ecto-nucleotidase E-NPP1

Glioblastomas are highly aggressive brain tumours and are characterised by substantial cellular heterogeneity within a single tumour. A sub-population of glioblastoma stem-like cells (GSCs) that shares properties with neural precursor cells has been described, exhibiting resistance to therapy and therefore being considered responsible for the high recurrence rate in glioblastoma. To elucidate the underlying cellular processes we investigated the role of phosphatases in the GSC phenotype, using an in vitro phosphatome-wide RNA interference screen. We identified a set of genes, the knockdown of which induces a significant decrease in the glioma stem cell marker CD133, indicating a role in the glioblastoma stem-like phenotype. Among these genes, the ecto-nucleotidase ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) was found to be highly expressed in GSCs compared with normal brain and neural stem cells. Knockdown of ENPP1 in cultured GSCs resulted in an overall downregulation of stem cell-associated genes, induction of differentiation into astrocytic cell lineage, impairment of sphere formation, in addition to increased cell death, accumulation of cells in G1/G0 cell cycle phase and sensitisation to chemotherapeutic treatment. Genome-wide gene expression analysis and nucleoside and nucleotide profiling revealed that knockdown of ENPP1 affects purine and pyrimidine metabolism, suggesting a link between ENPP1 expression and a balanced nucleoside-nucleotide pool in GSCs. The phenotypic changes in E-NPP1-deficient GSCs are assumed to be a consequence of decreased transcriptional function of E2F1. Together, these results reveal that E-NPP1, by acting upstream of E2F1, is indispensable for the maintenance of GSCs in vitro and hence required to keep GSCs in an undifferentiated, proliferative state.

Role of GALNT2 in the modulation of ENPP1 expression, and insulin signaling and action: GALNT2: A novel modulator of insulin signaling

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling and action. Understanding the mechanisms underlying ENPP1 expression may help unravel molecular mechanisms of insulin resistance. Recent data suggest a role of ENPP1-3′untraslated region (UTR), in controlling ENPP1 expression. We sought to identify trans-acting ENPP1-3′UTR binding proteins, and investigate their role on insulin signaling.By RNA pull-down, 49 proteins bound to ENPP1-3′UTR RNA were identified by mass spectrometry (MS). Among these, in silico analysis of genome wide association studies and expression profile datasets pointed to N-acetylgalactosaminyltransferase 2 gene (GALNT2) for subsequent investigations. Gene expression levels were evaluated by RT-PCR. Protein expression levels, IRS-1 and Akt phosphorylation were evaluated by Western blot. Insulin receptor (IR) autophosphorylation was evaluated by ELISA.GALNT2 down-regulation increased while GALNT2 over-expression reduced ENPP1 expression levels. In addition, GALNT2 down-regulation reduced insulin stimulation of IR, IRS-1 and Akt phosphorylation and insulin inhibition of phosphoenolpyruvate carboxykinase (PEPCK) expression, a key neoglucogenetic enzyme.Our data point to GALNT2 as a novel factor involved in the modulation of ENPP1 expression as well as insulin signaling and action in human liver HepG2 cells.

Intermittent Cyclic Mechanical Tension‐induced Down‐regulation of Ectonucleotide Pyrophosphatase Phosphodiesterase 1 Gene Expression is Mainly Dependent on TGF‐β1 in End‐plate Chondrocytes

To investigate the relationship between ectonucleotide pyrophosphatase phosphodiesterase-1(ENPP-1) expression and transforming growth factor beta 1 (TGF-β1) of end-plate chondrocytes after stimulation with intermittent cyclic mechanical tension (ICMT) by using an FX-4000T Flexercell Tension Plus unit. Rat end-plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) applied for 7 days for 4 h/day and cultured for a further 2 days. End-plate chondrocytes were also exposed to 10 ng/mL of TGF-β1. Then, using small interfering RNA technology, small interfering TGF-β1 (siTGF-β1) was transfected. Expression of ENPP-1 and TGF-β1 was measured by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and western blotting. Expression of both ENPP-1 and TGF-β1 was up-regulated after ICMT. Both RT-PCR and western blot showed that ENPP-1 expression decreases with siRNA TGF-β1 after 3% elongation 40 min, and cultured for an additional 2 days. It was found that down-regulation of ENPP-1 gene expression induced by ICMT is likely dependent on TGF-β1 in end-plate chondrocytes.

236: Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) expression in adipose tissue of women with excessive versus normal gestational weight gain during pregnancy

1 (ENPP1) expression in adipose tissue of women with excessive versus normal gestational weight gain during pregnancy Aaron Poole, Gayle Olson, Batbayar Tumurbaatar, Kathleen Vincent, Yongquan Jiang, Massoud Motamedi, Gracie Vargas, Manisha Chandalia, Abate Nicola University of Texas Medical Branch Galveston, Obstetrics & Gynecology, Galveston, TX, University of Texas Medical Branch Galveston, Internal Medicine–Endocrinology, Galveston, TX, University of Texas Medical Branch Galveston, Center for Biomedical Engineering, Galveston, TX OBJECTIVE: ENPP1, a transmembrane glycoprotein, has been shown to modulate adipocyte maturation and insulin receptor signaling. These effects have been associated with systemic insulin resistance and increased risk for type 2 diabetes. Our objective is to measure adipocyte ENPP1 expression in response to gestational weight gain (GWG). STUDY DESIGN: Women scheduled for elective repeat cesarean at term and who fasted at least 6 hours were recruited. Blood was obtained before the initiation of intravenous fluids and subcutaneous fat was biopsied after the skin incision. Adipose cell size was analyzed using 3D multi-photon imaging. Tissue expression level of ENPP1 and phosphorylation of Akt (pAkt) for insulin signaling were measured by Western Blot. Using IOM guidelines, excessive vs normal GWG were compared. Statistical Analysis Software was utilized. RESULTS: Fifteen subjects with excessive GWG were compared to 9 with normal GWG. Maternal age, EGA at delivery and pregestational body mass index (BMI) were not significantly different. Delivery BMI, birthweight, tissue expression level of ENPP1, adipocyte cell size and phosphorylated Akt were significantly different (Table). CONCLUSION: Increased expression level of ENPP1 in women with excessive GWG is associated with decreased adipocyte cell size and phosphorylation of Akt, indicating impaired maturation of adipocytes and insulin signaling. These findings suggest women with excessive GWG may be at risk for future systemic insulin resistance and type 2 diabetes. Sample characteristics–Estimated preterm birth prevalences by subgroup

The role of ecto-nucleotide pyrophosphatase/phosphodiesterase 1 in diabetic nephropathy

The increased prevalence of diabetes mellitus has caused a rise in the occurrence of its chronic complications, such as diabetic nephropathy (DN), which is associated with elevated morbidity and mortality. Familial aggregation studies have demonstrated that besides the known environmental risk factors, DN has a major genetic component. Therefore, it is necessary to identify genes associated with risk for or protection against DN. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is expressed in several tissues, including the kidneys. Increased levels of ENPP1 expression inhibit tyrosine-kinase activity of the insulin receptor in several cell types, leading to insulin resistance. K121Q polymorphism of the ENPP1 gene seems to be associated with insulin resistance and DN development. The elucidation of genetic factors and their associations will provide better understanding of the pathogenesis of DN and, may consequently, lead to a more effective approach to prevention and treatment.

Ectonucleotide Pyrophosphatase/Phosphodiesterase-1 (ENPP1) Protein Regulates Osteoblast Differentiation

ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase-1) is an established regulator of tissue mineralization. Previous studies demonstrated that ENPP1 is expressed in differentiated osteoblasts and that ENPP1 influences matrix mineralization by increasing extracellular levels of inorganic pyrophosphate. ENPP1 is also expressed in osteoblastic precursor cells when stimulated with FGF2, but the role of ENPP1 in preosteoblastic and other precursor cells is unknown. Here we investigate the function of ENPP1 in preosteoblasts. We find that ENPP1 expression is critical for osteoblastic differentiation and that this effect is not mediated by changes in extracellular concentration levels of phosphate or pyrophosphate or ENPP1 catalytic activity. MC3T3E1(C4) preosteoblastic cells, in which ENPP1 expression was suppressed by ENPP1-specific shRNA, and calvarial cells isolated from Enpp1 knock-out mice show defective osteoblastic differentiation upon stimulation with ascorbate, as indicated by a lack of cellular morphological change, a lack of osteoblast marker gene expression, and an inability to mineralize matrix. Additionally, MC3T3E1(C4) cells, in which wild type or catalytic inactive ENPP1 expression was increased, exhibited an increased tendency to differentiate, as evidenced by increased osteoblast marker gene expression and increased mineralization. Notably, treatment of cells with inorganic phosphate or pyrophosphate inhibited, as opposed to enhanced, expression of multiple genes that are expressed in association with osteoblast differentiation, matrix deposition, and mineralization. Our results indicate that ENPP1 plays multiple and distinct roles in the development of mineralized tissues and that the influence of ENPP1 on osteoblast differentiation and gene expression may include a mechanism that is independent of its catalytic activity.

The role of HSP70 on ENPP1 expression and insulin-receptor activation

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin-receptor (IR) signaling and, when over-expressed, induces insulin resistance in vitro and in vivo. Understanding the regulation of ENPP1 expression may, thus, unravel new molecular mechanisms of insulin resistance. Recent data point to a pivotal role of the ENPP1 3'UTR, in modulating ENPP1 mRNA stability and expression. We sought to identify trans-acting proteins binding the ENPP1-3'UTR and to investigate their role on ENPP1 expression and on IR signaling. By RNA electrophoresis mobility shift analysis and tandem mass spectrometry, we demonstrated the binding of heat shock protein 70 (HSP70) to ENPP1-3'UTR. Through this binding, HSP70 stabilizes ENPP1 mRNA and increases ENPP1 transcript and protein levels. This positive modulation of ENPP1 expression is paralleled by a reduced insulin-induced IR and IRS-1 phosphorylation. Taken together these data suggest that HSP70, by affecting ENPP1 expression, may be a novel mediator of altered insulin signaling.

Hepatic ENPP1 expression is induced in diabetic rabbits

The ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an inhibitor of the insulin receptor. Variants of ENPP1 are associated with infantile arterial calcification, obesity, and insulin resistance. To study the functional relevance of this protein in vivo, we cloned rabbit ENPP1 and studied its regulation in experimentally induced diabetes mellitus. We amplified and sequenced the complete coding sequence of rabbit ENPP1 gene out of a liver cDNA library using redundant primers deduced from other species. Next, we performed quantitative PCR of ENPP1 to study the tissue distribution of ENPP1 expression and its regulation in an alloxan-dependent diabetes model. The putative rabbit ENPP1 protein contains 873 amino acids and is highly conserved when compared with human ENPP1 (90% amino acid identity). Particularly high levels of ENPP1 mRNA expression were found in adipose tissue. Quantitative PCR analysis revealed a significant upregulation of ENPP1 transcription in liver (p = 0.025) and brain (p = 0.034) of diabetic rabbits compared with controls. Hepatic ENPP1 expression is induced in diabetic rabbits when compared with controls. This approximately twofold upregulation of ENPP1 mRNA in rabbit liver parallels previous findings in patients with type 2 diabetes mellitus. We provide further molecular information about ENPP1 as a potential pharmacologic target and characterize its regulation in an insulin-dependent diabetes mellitus animal model.