The POLD1R689W variant increases the sensitivity of colorectal cancer cells to ATR and CHK1 inhibitors.

Inhibition of the kinase ATR, a central regulator of the DNA damage response, eliminates subsets of cancer cells in certain tumors. As previously shown, this is at least partly attributable to synthetic lethal interactions between ATR and POLD1, the catalytic subunit of the polymerase δ. Various POLD1 variants have been found in colorectal cancer, but their significance as therapeutic targets for ATR pathway inhibition remains unknown. Using CRISPR/Cas9 in the colorectal cancer cell line DLD-1, which harbors four POLD1 variants, we established heterozygous POLD1-knockout clones with exclusive expression of distinct variants to determine the functional relevance of these variants individually by assessing their impact on ATR pathway activation, DNA replication, and cellular sensitivity to inhibition of ATR or its effector kinase CHK1. Of the four variants analyzed, only POLD1R689W affected POLD1 function, as demonstrated by compensatory ATR pathway activation and impaired DNA replication. Upon treatment with ATR or CHK1 inhibitors, POLD1R689W strongly decreased cell survival in vitro, which was attributable at least partly to S phase impairment and apoptosis. Similarly, treatment with the ATR inhibitor AZD6738 inhibited growth of murine xenograft tumors, harboring the POLD1R689W variant, in vivo. Our POLD1-knockout model thus complements algorithm-based models to predict the pathogenicity of tumor-specific variants of unknown significance and illustrates a novel and potentially clinically relevant therapeutic approach using ATR/CHK1 inhibitors in POLD1-deficient tumors.

Fetuin-B, a liver-derived plasma protein is essential for fertilization.

The zona pellucida (ZP) is a glycoprotein matrix surrounding mammalian oocytes. Upon fertilization, ZP hardening prevents sperm from binding to and penetrating the ZP. Here, we report that targeted gene deletion of the liver-derived plasma protein fetuin-B causes premature ZP hardening and, consequently, female infertility. Transplanting fetuin-B-deficient ovaries into wild-type recipients restores fertility, indicating that plasma fetuin-B is necessary and sufficient for fertilization. In vitro fertilization of oocytes from fetuin-B-deficient mice only worked after rendering the ZP penetrable by laser perforation. Mechanistically, fetuin-B sustains fertility by inhibiting ovastacin, a cortical granula protease known to trigger ZP hardening. Thus, plasma fetuin-B is necessary to restrain protease activity and thereby maintain ZP permeability until after gamete fusion. These results also show that premature ZP hardening can cause infertility in mice.

Accelerated growth plate mineralization and foreshortened proximal limb bones in fetuin-A knockout mice.

The plasma protein fetuin-A/alpha2-HS-glycoprotein (genetic symbol Ahsg) is a systemic inhibitor of extraskeletal mineralization, which is best underscored by the excessive mineral deposition found in various tissues of fetuin-A deficient mice on the calcification-prone genetic background DBA/2. Fetuin-A is known to accumulate in the bone matrix thus an effect of fetuin-A on skeletal mineralization is expected. We examined the bones of fetuin-A deficient mice maintained on a C57BL/6 genetic background to avoid bone disease secondary to renal calcification. Here, we show that fetuin-A deficient mice display normal trabecular bone mass in the spine, but increased cortical thickness in the femur. Bone material properties, as well as mineral and collagen characteristics of cortical bone were unaffected by the absence of fetuin-A. In contrast, the long bones especially proximal limb bones were severely stunted in fetuin-A deficient mice compared to wildtype littermates, resulting in increased biomechanical stability of fetuin-A deficient femora in three-point-bending tests. Elevated backscattered electron signal intensities reflected an increased mineral content in the growth plates of fetuin-A deficient long bones, corroborating its physiological role as an inhibitor of excessive mineralization in the growth plate cartilage matrix--a site of vigorous physiological mineralization. We show that in the case of fetuin-A deficiency, active mineralization inhibition is a necessity for proper long bone growth.

Clearance of fetuin-A--containing calciprotein particles is mediated by scavenger receptor-A.

RATIONALE: Fetuin-A is a liver-derived plasma protein involved in the regulation of calcified matrix metabolism. Biochemical studies showed that fetuin-A is essential for the formation of protein-mineral complexes, called calciprotein particles (CPPs). CPPs must be cleared from circulation to prevent local deposition and pathological calcification. OBJECTIVE: We studied CPP clearance in mice and in cell culture to identify the tissues, cells, and receptors involved in the clearance. METHODS AND RESULTS: In mice, fetuin-A-containing CPPs were rapidly cleared by the reticuloendothelial system, namely Kupffer cells of the liver and marginal zone macrophages of the spleen. Macrophages from scavenger receptor-AI/II (SR-A)-deficient mice cleared CPPs less efficiently than macrophages from wild-type mice, suggesting that SR-AI/II is involved in CPP binding and endocytosis. Accordingly, we found reduced clearance of CPPs in SR-A/MARCO-deficient mice. CONCLUSIONS: We could demonstrate that fetuin-A-containing CPPs facilitate the clearance of mineral debris by macrophages via SR-A. Since the same receptor also contributes to the uptake of modified low-density lipoprotein particles in atherosclerosis, defective endocytosis of both types of particle may impinge on lipid as well as mineral debris clearance in calcifying atherosclerosis.

Fetuin-A regulation of calcified matrix metabolism.

The final step of biomineralization is a chemical precipitation reaction that occurs spontaneously in supersaturated or metastable salt solutions. Genetic programs direct precursor cells into a mineralization-competent state in physiological bone formation (osteogenesis) and in pathological mineralization (ectopic mineralization or calcification). Therefore, all tissues not meant to mineralize must be actively protected against chance precipitation of mineral. Fetuin-A is a liver-derived blood protein that acts as a potent inhibitor of ectopic mineralization. Monomeric fetuin-A protein binds small clusters of calcium and phosphate. This interaction results in the formation of prenucleation cluster-laden fetuin-A monomers, calciprotein monomers, and considerably larger aggregates of protein and mineral calciprotein particles. Both monomeric and aggregate forms of fetuin-A mineral accrue acidic plasma protein including albumin, thus stabilizing supersaturated and metastable mineral ion solutions as colloids. Hence, fetuin-A is a mineral carrier protein and a systemic inhibitor of pathological mineralization complementing local inhibitors that act in a cell-restricted or tissue-restricted fashion. Fetuin-A deficiency is associated with soft tissue calcification in mice and humans.

The physiologic development of fetuin-a serum concentrations in children.

Fetuin-A prevents tissue calcification by forming soluble complexes with calcium and phosphate. A pathological depletion of serum fetuin-A has been observed in children on dialysis or after renal transplantation but knowledge on physiologic age-related changes in serum fetuin-A is limited. We prospectively evaluated serum fetuin-A in 133 infants and children, ranging from very low birth weight infants to adolescents. Highest serum fetuin-A levels were present between 23 and 30 wk of gestation (1 +/- 0.33 mg/mL). Thereafter, the values decreased. This decrease was linked to biological rather than chronological age. At 32 to 36 and 37 to 40 wk of gestation, the serum fetuin-A concentration was 0.63 +/- 0.26 and 0.63 +/- 0.21 mg/mL, respectively. Thereafter, the concentrations remained stable until adolescence at 0.58 +/- 0.12 mg/mL. Intercurrent infections were associated with a transient decrease of serum fetuin-A levels. The high serum fetuin-A concentrations in preterm children suggest that fetuin-A is of high physiologic impact for the fetal and the preterm-born organism, showing extensive tissue formation. This might point to a new mechanism contributing to organ damage in these patients, comparable with children on dialysis.

Fetuin-A protects against atherosclerotic calcification in CKD.

Reduced serum levels of the calcification inhibitor fetuin-A associate with increased cardiovascular mortality in dialysis patients. Fetuin-A-deficient mice display calcification of various tissues but notably not of the vasculature. This absence of vascular calcification may result from the protection of an intact endothelium, which becomes severely compromised in the setting of atherosclerosis. To test this hypothesis, we generated fetuin-A/apolipoprotein E (ApoE)-deficient mice and compared them with ApoE-deficient and wild-type mice with regard to atheroma formation and extraosseous calcification. We assigned mice to three treatment groups for 9 wk: (1) Standard diet, (2) high-phosphate diet, or (3) unilateral nephrectomy (causing chronic kidney disease [CKD]) plus high-phosphate diet. Serum urea, phosphate, and parathyroid hormone levels were similar in all genotypes after the interventions. Fetuin-A deficiency did not affect the extent of aortic lipid deposition, neointima formation, and coronary sclerosis observed with ApoE deficiency, but the combination of fetuin-A deficiency, hyperphosphatemia, and CKD led to a 15-fold increase in vascular calcification in this model of atherosclerosis. Fetuin-A deficiency almost exclusively promoted intimal rather than medial calcification of atheromatous lesions. High-phosphate diet and CKD also led to an increase in valvular calcification and aorta-associated apoptosis, with wild-type mice having the least, ApoE-deficient mice intermediate, and fetuin-A/ApoE-deficient mice the most. In addition, the combination of fetuin-A deficiency, high-phosphate diet, and CKD in ApoE-deficient mice greatly enhanced myocardial calcification, whereas the absence of fetuin-A did not affect the incidence of renal calcification. In conclusion, fetuin-A inhibits pathologic calcification in both the soft tissue and vasculature, even in the setting of atherosclerosis.

Hierarchical role of fetuin-A and acidic serum proteins in the formation and stabilization of calcium phosphate particles.

The serum protein fetuin-A is a potent systemic inhibitor of soft tissue calcification. Fetuin-A is highly effective in the formation and stabilization of protein-mineral colloids, referred to as calciprotein particles (CPPs). These particles ripen in vitro in a two-step process, indicated by a morphological conversion from spheres to larger prolate ellipsoids. Using a combined light scattering and electron microscopic imaging approach we determined that the second-stage particles resulted from a highly anisotropic outgrowth of the first-stage particles. Electron microscopy of ascites fluid from a patient with calcifying peritonitis revealed particles reminiscent of secondary CPPs. Thus, CPPs form in the body and undergo the two-step ripening at least in pathological conditions. Unlike in vitro generated CPPs, ascites-derived CPPs contained little fetuin-A but large amounts of albumin. This prompted us to study the role of fetuin-A combined with other serum proteins in CPP formation. Fetuin-A was indispensable for primary CPP formation. Albumin and acidic proteins in general greatly enhanced the fetuin-A triggered formation of secondary CPPs and, thus, substituted substantial amounts of fetuin-A without loss of inhibition of calcium phosphate precipitation. Thus, direct mineral deposition from solute in the body is unlikely even at low fetuin-A serum levels as long as sufficient bulk acidic protein is available. Collectively fetuin-A and other acidic bulk plasma proteins may be considered as mineral chaperones mediating the stabilization, safe transport, and clearance in the body of calcium and phosphate as colloidal complexes, thus, preventing ectopic calcification.

Mineral chaperones: a role for fetuin-A and osteopontin in the inhibition and regression of pathologic calcification.

Clinical nephrologists are well aware of the consequences of pathologic mineralization (calcification). Several studies have found a strong association between vascular and valvular mineralization and advanced or end-stage chronic kidney disease (CKD), with shorter survival times and increased morbidity. In the cardiology community, until quite recently, ectopic mineralization was considered harmless or even beneficial. Some still assume that atherosclerotic intima mineralization stabilizes atherosclerotic plaques, thus doing more good than harm. We suggest that vascular mineralization and indeed soft tissue mineralization in general may be a way in which the body deals with certain adverse situations involving local inflammation, associated tissue damage and tissue remodeling. Ectopic soft tissue mineralization resembles physiological bone mineralization in many ways. Markers of mineralizing bone also are present during soft tissue mineralization. We postulate that it may be possible to reverse soft tissue mineralization by applying selected principles of bone catabolism, namely mineral dissolution and phagocytosis. We consider putative strategies for therapeutic intervention to maximize the clearing of calcified debris particles. In particular, we discuss the roles of the plasma protein fetuin-A/alpha2HS-glycoprotein and the mineral-binding protein osteopontin in the prevention and possible regression of mineralization in disease.

Fetuin-A (AHSG) prevents extraosseous calcification induced by uraemia and phosphate challenge in mice.

BACKGROUND: Chronic kidney disease (CKD) is associated with vascular and tissue calcification. The extent of vascular calcification has been identified as an independent risk factor of cardiovascular death in patients on haemodialysis. METHODS: We studied the role of fetuin-A in CKD-associated calcification using a mouse model of graded renal insufficiency generated by nephrectomy and high phosphate diet. We used wild-type and fetuin-A-deficient mice on the calcification resistant genetic background C57BL/6 to study the influence on calcification of CKD, dietary phosphate and fetuin deficiency. Hyperphosphataemia, elevated BUN, hyperparathyroidism and von Kossa histochemistry served as indicators of calcification disease. The expression of osteopontin, a marker of osteoblast-like cell differentiation was analyzed by realtime PCR and immunohistochemistry. RESULTS: We detected tissue and genotype-specific susceptibility for calcification. Fetuin-A-deficient mice with CKD and high phosphate diet had only a moderately elevated serum calcium phosphate product (6.9 +/- 1.4 mmol(2)/l(2)), but suffered severe calcification of kidney, heart and lung. In contrast, wild-type mice under the same conditions developed renal calcinosis only despite an elevated serum calcium phosphate product (9.6 +/- 0.9 mmol(2)/l(2)). Calcification was preceded by the local induction of osteopontin, a marker for osteoblast-like cell differentiation. CONCLUSION: Fetuin-A deficiency, CKD and high phosphate diet act synergistically in the pathogenesis of extraosseous calcification.

Myocardial stiffness, cardiac remodeling, and diastolic dysfunction in calcification-prone fetuin-A-deficient mice.

Accelerated atherosclerosis in dialysis patients is characterized by severe vascular calcification, and the magnitude of vascular calcification is associated with increased cardiovascular mortality. Calcification-dependent arterial stiffness is considered to be a major determinant of cardiac failure in uremia. Fetuin-A/alpha(2)-Heremans-Schmid glycoprotein is an abundant serum protein with powerful calcification inhibitory properties. Fetuin-A deficiency was recently linked to cardiovascular mortality in dialysis patients. Fetuin-A knockout (fetuin-KO) mice spontaneously develop widespread soft tissue calcification, including significant myocardial calcification, whereas larger arteries are spared. Therefore, this investigation offers the unique opportunity to study the functional role of isolated myocardial calcification independent of arterial stiffness by assessing the hemodynamics of fetuin-KO mice. Cardiac output in fetuin-KO mice was lower than in wild-type mice (fetuin-KO 1.81 +/- 0.18 versus WT 2.45 +/- 0.29 ml/min per g; P < 0.005), and fetuin-KO mice were refractory to dobutamine stimulation. Left ventricular relaxation was significantly impaired in fetuin-KO hearts with the relaxation index reduced by 23% (P < 0.005). After ischemia, fetuin-KO hearts displayed a continuous decline in left ventricular developed pressure after the initial phase of reperfusion, resulting in 77 +/- 15% of preischemic left ventricular developed pressure (P < 0.05 versus wild-type). In fetuin-KO mice, dystrophic cardiac calcification, with myocardial calcium contents increased 60-fold, was associated with profound induction of profibrotic TGF-beta and downstream collagen and fibronectin mRNA synthesis. In conclusion, independent of arterial stiffness, calcification-associated "myocardial stiffness" characterized by cardiac fibrosis, diastolic dysfunction, impaired tolerance to ischemia, and catecholamine resistance thus may constitute an underestimated cardiovascular risk factor that contributes to cardiac failure in calcification-prone states.

Tissue distribution and activity testing suggest a similar but not identical function of fetuin-B and fetuin-A.

Fetuins are serum proteins with diverse functions including the regulation of osteogenesis and inhibition of unwanted mineralization. Besides the alpha2-Heremans and Schmid glycoprotein/fetuin-A, the recently identified fetuin-B is a second member of the fetuin family [Olivier, Soury, Risler, Smih, Schneider, Lochner, Jouzeau, Fey and Salier (1999) Genomics 57, 352-364; Olivier, Soury, Ruminy, Husson, Parmentier, Daveau and Salier (2000) Biochem. J. 350, 589-597], which belongs to the cystatin superfamily. We compared the expressions of fetuin-B and fetuin-A at the RNA level and established that both genes are most highly expressed in liver tissue. Like fetuin-A, fetuin-B mRNA is also highly expressed in tongue and placenta tissues. We demonstrated for the first time that fetuin-B is also expressed at the protein level in sera and several organs of mouse, rat and human. We isolated contiguous genomic clones containing both fetuin-B and fetuin-A genes, indicating that these genes are closely linked at the genome level. The close proximity of both these genes may explain our observation that fetuin-B expression was decreased in fetuin-A-deficient mice. Unlike fetuin-A, the amount of fetuin-B protein in human serum varied with gender and was higher in females than in males. Functional analysis revealed that fetuin-B, similarly to fetuin-A, is an inhibitor of basic calcium phosphate precipitation, albeit less active when compared with fetuin-A. Therefore fetuin-B may have a function that is partly overlapping, if not identical, with the function of fetuin-A.

The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification.

Ectopic calcification is a frequent complication of many degenerative diseases. Here we identify the serum protein alpha2-Heremans-Schmid glycoprotein (Ahsg, also known as fetuin-A) as an important inhibitor of ectopic calcification acting on the systemic level. Ahsg-deficient mice are phenotypically normal, but develop severe calcification of various organs on a mineral and vitamin D-rich diet and on a normal diet when the deficiency is combined with a DBA/2 genetic background. This phenotype is not associated with apparent changes in calcium and phosphate homeostasis, but with a decreased inhibitory activity of the Ahsg-deficient extracellular fluid on mineral formation. The same underlying principle may contribute to many calcifying disorders including calciphylaxis, a syndrome of severe systemic calcification in patients with chronic renal failure. Taken together, our data demonstrate a critical role of Ahsg as an inhibitor of unwanted mineralization and provide a novel therapeutic concept to prevent ectopic calcification accompanying various diseases.