ENPP1 in Blood and Bone: Skeletal and Soft Tissue Diseases Induced by ENPP1 Deficiency

Abstract

The enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein that hydrolyzes extracellular ATP to generate pyrophosphate (PP_i) and adenosine monophosphate, thereby contributing to downstream purinergic signaling pathways. The clinical phenotypes induced by ENPP1 deficiency are seemingly contradictory and include early-onset osteoporosis in middle-aged adults and life-threatening vascular calcifications in the large arteries of infants with generalized arterial calcification of infancy. The progressive overmineralization of soft tissue and concurrent undermineralization of skeleton also occur in the general medical population, where it is referred to as paradoxical mineralization to highlight the confusing pathophysiology. This review summarizes the clinical presentation and pathophysiology of paradoxical mineralization unveiled by ENPP1 deficiency and the bench-to-bedside development of a novel ENPP1 biologics designed to treat mineralization disorders in the rare disease and general medical population.

Keywords: ARHR2; DISH; ENPP1 deficiency; GACI; OPLL; PXE; autosomal recessive hypophosphatemic rickets type 2; dystrophic idiopathic spinal hyperostosis; ectonucleotide pyrophosphatase/phosphodiesterase 1; enthesopathy; generalized arterial calcification of infancy; ossification of the posterior longitudinal ligament; pseudoxanthoma elasticum.

Figure 1

Metabolic diseases due to homozygous defects in extracellular purinergic metabolism. The liver-associated transmembrane transporter ABCC6 [adenosine triphosphate (ATP)-binding cassette subfamily C member 6] exports ATP into the extracellular space, which is then metabolized by ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) into adenosine monophosphate (AMP) and pyrophosphate (PP_i). AMP is metabolized into adenosine and phosphate (P_i) by 5′-nucleotidase (CD73), and PP_i is metabolized into P_i by tissue-nonspecific alkaline phosphatase (TNAP). P_i promotes, and PP_i inhibits, hydroxyapatite, which constitutes the hard matrix of bone and ectopic calcifications in soft tissue. Pathogenic variants in ENPP1 and ABCC6 reduce extracellular plasma (PP_i) to 10% and 30% of normal, respectively, inducing generalized calcification of infancy (GACI), in which approximately 50% of afflicted infants will succumb to life-threatening aortic and arterial calcifications. Those that survive will almost invariably exhibit phosphate wasting rickets in childhood, called autosomal recessive hypophosphatemic rickets type 2 (ARHR2).

Figure 2

Skeletal phenotype of a 15-year-old autosomal recessive hyperphosphatemic rickets type 2 (ARHR2) patient before and after surgical correction and phosphate supplementation. (a) Prominent skeletal deformities with bending of long bones in lower limb were corrected by bilateral hemiepiphysiodesis, (b) requiring open reduction and internal fixation by a locking compression plate and traumatic diaphyseal femur fracture. (c) Radiograph of left hand and distal forearm of the patient at 14 years of age with fraying, spraying, and cupping of the metaphysis of the distal radius and ulna, which (d) was observed to improve following 2 years of supplemental phosphate and calcitriol therapy. The institution of supplemental phosphate and calcitriol (dashed line in panels e and f) reduced c-terminal fibroblast growth factor 23 (cFGF23) in plasma [measured in kilo–relative units per liter (kRU/L)] (e) and maintained or increased serum phosphate (f), but bone mass continued to decrease in the hip (g) and total body less head (TBLH) (h), as quantitated by the height-adjusted Z-score (HAZ). (i) Computed tomography of the abdomen of an ARHR2 child (aged 8 years, 3 months) treated with supplemental calcitriol and phosphate revealing bilateral calcification of the renal pyramids. (j) Renal histology of a deceased generalized arterial calcification of infancy (GACI) infant revealing foci of calcifications within the renal cortex. Figure adapted from Reference .

Figure 3

Spinal and Achilles enthesopathies in diffuse idiopathic skeletal hyperostosis patients with ENPP1 haploinsufficiency. (a) Multiple paraspinal ligament ossifications (open arrowheads) and compression fractures (white arrows) in a patient with a heterozygous ENPP1 Y451C pathogenic variant. (b) Multiple compression fractures in the spine of a second patient with a heterozygous ENPP1 N179S pathogenic variant (blue arrows). (c) Pedigree of a patient (black arrow) with compound ENPP1 pathogenic variants, one of which (Y451C) was passed on to both sons (asterisks), who both exhibited Achilles tendon enthesis calcification at the ages of 19 and 23 years. (d) Schematic of ENPP1 illustrating the location of variants (in SMB2 and the catalytic domain). The nuclease domain is omitted to conserve space. (e) When compared with WT ENPP1, the N179S and Y451C variants reduced activity by 55% and 70%, respectively (**** indicates p < 0.0001). Abbreviations: CD, cytoplasmic domain; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; SMB, somatomedin B-like domain; TM, transmembrane domain; WT, wild type. Figure adapted from Reference .

Figure 4

ENPP1 deficiency and low mammalian bone mass. (a) Superposition of Cα traces of human ENPP4 (PDB code 4LQY) with mouse ENPP1 (PDB code 4B56), mouse ENPP2 (PDB code 3NKN), and a bacterial ENPP (PDB code 2GSU) to illustrate the degree of structural conservation of the catalytic domain (with an average RMSD of 0.68 Å), which includes the active site zinc ions, the α-helix on which the catalytic threonine is located, and the backbone near the hydrophobic slot. Regions of moderate similarity include the second shell of the active site (average RMSD of 1.33 Å), and the lowest similarity is the substrate binding domain of the active site (average RMSD of 2.51 Å). The ccp4 program Superpose was used to overlay the conserved subdomain (124). Active site zinc ions are depicted as spheres. (b) Pedigree of a family possessing individuals with homozygous and heterozygous ENPP1 Y451C pathogenic variants. (c) Plasma PP_i levels of the family members plotted by their mutational status. (d) Micro-CT image of thoracic spine in a patient with an ENPP1 heterozygous Y451C pathogenic variant revealing multiple compression fractures. (e) Areal bone mineral density T-scores in a patient with ARHR2, displayed as box plots denoting median value and interquartile range. Abbreviations: AP, anteroposterior; ARHR2, autosomal recessive hyperphosphatemic rickets type 2; BMD, bone mineral density; micro-CT, microcomputed tomography; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; PDB, Protein Data Bank; PP_i, pyrophosphate; RMSD, root mean square deviation; WT, wild type. Figure adapted from References and .

Figure 5

Differential gene expression in the tibias of 10-week-old male Enpp1^asj and WT mice analyzed by RNA sequencing, (a) Volcano plots to illustrate the differential gene expression in 10-week-old male Enpp1^asj mice and WT siblings reveal the most upregulated transcript to be FGF23. (b) Transcriptome analysis of bone mineralization pathways disrupted by murine Enpp1 deficiency reveals suppression of Wnt due to increased expression of the Wnt inhibitors Wif1 in kidney and Sfrp1 in liver and decreased transcription of Wnt ligand Fzd8 in liver. Increased FGF23 in bone further inhibits bone mineralization through inhibition of trabecular bone (125) and suppression of Wnt (126). Abbreviations: ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; FGF23, fibroblast growth factor 23; WT, wild type. Figure adapted from Reference .

Figure 6

The T238A mouse. (a) Plasma PP_i was absent in both Enpp1^T238A and Enpp1^asj mice, demonstrating abrogation of ENPP1 catalytic activity in both genotypes. (b) Western blot of primary osteocytes derived from calvaria cells confirms comparable ENPP1 protein expression in T238A and WT mice and reduced expression in ASJ mice (normalized to GAPDH). Analysis of skeletal mineralization demonstrated preservation of trabecular (c), and cortical (d) mineralization parameters as well as biomechanical performance (e) in the tibias and femurs of Enpp1^T238A mice, as compared with Enpp1^asj mice. The data in panels c–e are displayed as change (Δ) relative to WT siblings using box plots to display median values and interquartile range. Superimposed on the box plots in green is the relative mean (bar) and standard error of the mean (whiskers) for each measurement. (f) Calvaria cells derived from WT, Enpp1^asj, and Enpp1^T238A mice and differentiated into osteocytes in a phosphate source for 21 days exhibit dramatic reductions in calcium phosphate in the Enpp1^asj mice (stain, Alizarin Red). (g) Quantitation of mRNA expression of Sfrp1 in calvaria osteoblasts derived from WT mice (black symbols) and Enpp1^asj mice (gray symbols) reveals elevations of Sfrp1 at days 7 and 21. Expression normalized to Hprt1. (h) Western blot of Sfrp1 knockout in Enpp1^asj calvaria cells demonstrates increased nuclear β-catenin, demonstrating that suppression of β-catenin signaling via SFRP1 accounts for the mineralization defect in ENPP1 deficiency. Values of p are explicitly stated as 0.05 ≥ p ≥ 0.001; *** indicates p < 0.001 and **** indicates p < 0.0001, Student’s unpaired t test (to respective WT sibling pairs). Abbreviations: ASJ, associated with stiff joints; BV/TV, bone volume to total volume fraction; Ct.Th, cortical thickness; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PP_i, pyrophosphate; Tb.N, trabecular number; Tb.Th, trabecular thickness; WT, wild type. Figure adapted from Reference .

Figure 7

ENPP1 regulates organismal physiology though catalysis-dependent and -independent mechanisms. ENPP1 catalytic activity regulates whole organismal physiology by generating catalysis-dependent products (in red) or hydrolyzing bioactive substrates (in green). Catalysis-independent effects of ENPP1 are mediated through ENPP1 protein signaling and are related to a commonly occurring polymorphism in the ENPP1 somatomedin B-like domain 2 (SMB2) domain—K121Q—identified by GWAS to be associated with childhood obesity (127) and increase risk of stroke in SCA (128). Finally, ENPP1 catalysis-independent pathways regulating bone mass have been recently confirmed in animal models (102). Red type is used to label catalytic products, green type is used to label catalytic substrates of ENPP1 regulating the listed phenotypes, and blue type is used to label protein mediators regulating the listed phenotypes in the figure. Abbreviations: cAMP, cyclic adenosine monophosphate; cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; ENPP1, ectonucleotide pyrophosphatase/phosphodiesterase 1; FGF23, fibroblast growth factor 23; GWAS, genome-wide association studies; PP_i, pyrophosphate; SCA, sickle cell anemia.

Figure 8

The efficacy of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) enzyme replacement in a murine model of generalized arterial calcification of infancy: biologic design, survival, and biomarkers. (a) Design of the first-generation murine Enpp1-Fc therapeutic. To produce a soluble recombinant protein, a segment of the extracellular region of NPP2 containing a furin cleavage site was substituted into ENPP1, and the Fc portion of immunoglobulin G1 (IgG1) was appended to the C-terminus. (b) The mean daily weights and standard deviations of wild-type (WT) Enpp1^WT (squares, n = 8), Enpp1-Fc-treated Enpp1^asj (triangles, n = 8), and vehicle-treated Enpp1^asj mice (circles, n = 8). Dosing began on day 14. Treatment consisted of daily (qD) doses of 10 mg/kg (or 500 au/kg) of Enpp1-Fc formulated in phosphate-buffered saline (PBS) and weekly injections of GK1.5. Vehicle consisted of PBS supplemented with zinc and calcium. Deaths in the untreated Enpp1^asj cohort are denoted by red arrows. No deaths occurred in Enpp1^WT or the treated Enpp1^asj cohort. (c) Survival curves for Enpp1-Fc (solid red line) and vehicle-treated (dashed black line) Enpp1^asj mice; p = 0.003 (Mantle-Cox). (d) Plasma pyrophosphate (PP_i) in Enpp1^WT and treated and untreated Enpp1^asj mice. The statistical significance between treated and untreated Enpp1^asj mice was p = 0.0015, Student’s two-tailed t test. Figure adapted from Reference .

Figure 9

The efficacy of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) enzyme replacement in a murine model of generalized arterial calcification of infancy: histology. (a) Postmortem high-resolution microcomputed tomography scans revealed extensive calcifications in untreated Enpp1^asj mice in the hearts, coronary arteries, and ascending and descending aortas but absolutely no calcifications in these organs in the treated Enpp1^asj cohort or in Enpp1^WT mice. (b) Untreated Enpp1^asj mice, right ventricle [hematoxylin and eosin (H&E) stain]. Two untreated Enpp1^asj mice had large, confluent, myocardial infarctions in the free wall of the right ventricle. All treated Enpp1^asj mice displayed normal right ventricle myocardium (not shown). (c) Untreated Enpp1^asj mice, right ventricle (H&E), showing detail from the boxed area in panel b. (d) Untreated Enpp1^asj mice, coronary arteries (H&E). All untreated Enpp1^asj mice had coronary calcifications, with most displaying circumferential calcifications in coronary arteries surrounded by scar tissue. (e) Untreated Enpp1^asj mice, myocardial septum (H&E). Nearly all animals (77%) displayed intracardiac calcifications surrounded by scar tissue, as demonstrated in this animal in the myocardial septum. Figure adapted from Reference .

Figure 10

The effects of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) enzyme replacement on skeletal mineralization. (a) Proximal tibia histology of 5-week-old wild-type (WT) mice and mice on the acceleration diet treated between weeks 3 and 5 with vehicle [phosphate-buffered saline (PBS)] or murine Enpp1-Fc. Morphologically apparent reductions in trabecular bone and markedly thinner growth plates are noted in vehicle-treated Enpp1^asj mice, and Enpp1-Fc treatment markedly improved trabecular bone volume and growth plate thickness. (b) Vehicle-treated Enpp1^asj mice on the acceleration diet exhibited pronounced nephrocalcinosis throughout the renal parenchyma, including cortex and renal medulla, in contrast to Enpp1-Fc-treated Enpp1^asj mice. The calcification was most pronounced in renal tubules near the cortical-medullary junction. Top and middle: hematoxylin and eosin (H&E) stains; bottom: Von Kossa stains. (c) Histomorphometry of mice demonstrated reductions in osteomalacia in the murine Enpp1-Fc treated cohorts; osteoid surface area per bone surface area (OS/BS), osteoblast surface per bone surface (ObS/BS), and osteoid volume per total volume (OV/TV) (female mice shown). (d) Trabecular mineralization as measured by microcomputed tomography, (e) biomechanical properties, and (f) length of femurs demonstrates recovery of long bone mineralization in the Enpp1-Fc-treated cohort. Trabecular bone volume to total volume fraction (BV/TV), trabecular number (Tb.N), biomechanical stiffness (slope of the load versus displacement curve), and max load (also known as strength) are shown as tested in three-point bending until failure in male and female mice; 14 WT mice were treated with PBS(5F and 9M), 9 Enpp1^asj mice were treated with PBS (4F and 5M), and 9 Enpp1^asj mice were treated with mEnpp1-Fc (4F and 5 M). (g) Nephrocalcinosis, quantitated by a renal pathologist blinded to experimental groups, yielded no statistical difference in nephrocalcinosis between the WT and Enpp1^asj mice treated with mEnpp1-Fc, while vehicle-treated Enpp1^asj mice experienced approximately a twofold increase in nephrocalcinosis when compared with WT or mEnpp1-Fc-treated Enpp1^asj mice. Individual measurements are displayed as circles, with bar height representing the median and error bars denoting the interquartile range (25%–75%); *** indicates p < 0.001 and **** indicates p < 0.0001 (analysis of variance). Figure adapted from Reference .

Figure 11

The efficacy of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) enzyme replacement in enthesopathy. (a) Representative histology of 23-week-old vehicle-treated wild-type (WT) and Enpp1^asj mice (top) compared with 23-week-old Enpp1^asj mice treated with weekly subcutaneous injections of 0.3 mg/kg BL-1118 chosen from complete responders, partial responders, and nonresponders (bottom). (b) Quantification of enthesis mineralization via Alizarin Red–stained sections in vehicle-treated WT and Enpp1^asj mice and BL-1118-treated Enpp1^asj mice. The data spread suggested grouping the response into complete responders, partial responders, and nonresponders (circled data points). (c) The same data as in panel b but grouped and analyzed into complete response (CR, 6 animals), partial response (PR, 7 animals), and nonresponse (NR, 6 animals). Black type and lines denote statistical significance between WT and treatment groups. Red type and lines denote statistical significance between treatment groups. Statistical significance is explicitly stated between 0.05 > p > 0.001; *** indicates p < 0.001 and **** indicates p < 0.0001 [analysis of variance (ANOVA) comparison of means]. Figure adapted from Reference .