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Regular Article |
From the Department of Pediatrics,*
Municipal
Children’s Hospital, Dortmund, Germany; the Department of
Medicine,
Veterans Affairs Medical Center,
University of California at San Diego, La Jolla, California; the
Department of Endocrinology,
Mount Zion
Hospital, University of California at San Francisco, San Francisco,
California; the Department of Pediatrics,§
Kobe
University School of Medicine, Kobe, Japan; the Department of
Immunology,
Scripps Research
Institute, La Jolla, California; and the University Children’s
Hospital,||
Zurich, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Calcification of the arterial ECM has been observed in certain human diseases (and in mouse models) in the absence of atherosclerosis, diabetes, renal failure, or a primary endocrine disorder affecting serum calcium or phosphate levels.1 In the human condition termed idiopathic infantile arterial calcification (IIAC), calcification (hydroxyapatite deposition) in the media of large muscular arteries is associated with a stenosing, fibroproliferative medial smooth muscle cell (SMC)-mediated process, maximal in the area of the internal elastic lamina.5-9
In the more than 160 cases of IIAC that have been reported, the disease most often appeared by early infancy, and was often lethal by 6 months of age, generally because of ischemic cardiomyopathy, and other complications of obstructive arteriopathy including renal artery stenosis.5-8 In more than a dozen reported cases of IIAC, peri-articular calcifications of large joints also developed in infancy.5-8
One theory for the basis of pathological arterial ECM calcification has proposed an active mineralization process mediated by factors including de novo generation by cells within the arterial wall of oxidized lipids, promineralizing matrix vesicles, and ectopically expressed osteoblastic noncollagenous proteins such as osteopontin.1,2 Alternatively, the physiological importance of genetically regulated inhibitors of calcification, ie, passive mineralization, has been illustrated in several human metabolic diseases (eg, hypoparathyroidism).
One of the major physiological inhibitors of calcification is PPi, which potently inhibits basic calcium phosphate (hydroxyapatite) crystal deposition in bone and cartilage.10,11 Significantly, markedly decreased plasma PPi9 and urinary PPi levels,8 respectively, have each been reported in individual case studies of IIAC. Some IIAC patients also have responded to treatment with bisphosphonates, which are nonhydrolyzable analogues of PPi.5,8
PPi is produced by a variety of biochemical reactions, and cells appear to channel intracellular PPi to the extracellular space.10-12 Reactions that generate PPi include the nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activities of a group of ecto-enzymes in the phosphodiesterase nucleotide pyrophosphatase (PDNP) family (EC 3.6.1.8, EC 3.1.4.1).13,14 PDNP family NTPPPH isozymes produce free PPi by hydrolysis of the phosphodiester-I bond in both purine and pyrimidine nucleoside triphosphates, and have been recognized to contribute to the regulation of intracellular and extracellular PPi levels in several tissues.10,12-14
PDNP family isozymes with NTPPPH activity are expressed as cell-bound class II (intracellular N-terminus) transmembrane glycoproteins of 120 to 130 kd that share a highly homologous extracellular domain containing two somatomedin B-like regions and a highly conserved catalytic site.13,14 Furthermore, soluble enzymatically active NTPPPH species are liberated by proteolysis of the parent molecules.13,14
The most widely distributed PDNP family NTPPPH isozyme is plasma cell membrane glycoprotein-1 (PC-1),10,12-14 which is particularly abundant in fibroblasts, chondrocytes, osteoblasts, and hepatocytes, and also circulates in soluble form(s) in plasma.15 The neural, enteric, and genitourinary tract PDNP family NTPPPH isozyme B10/PDNP3 also has been detected in a soluble form in plasma.16 PC-1 and B10/PDNP3 have nonhomologous cytosolic domains but highly homologous extracellular domains, and are likely to have risen from antecedent gene duplication, as their respective genes lie in close proximity on human chromosome 6q21-23.13,14
Another PDNP family NTPPPH isozyme, the alternatively spliced
autotaxin/PD1
, has been identified and demonstrated to be
particularly abundant in neural, intestinal, skin, and joint
tissues.13,14,17-19
Unlike PC-1 and B10/PDNP3, autotaxin
is predominantly released from cells via proteolysis,17,18
and autotaxin/PD-1 has both threonine type ATPase and NTPPPH
activities.19
Furthermore, unlike PC-1, forced expression
by transfection of autotaxin did not elevate extracellular PPi in
meniscal cells in vitro.20
Several cell types express more than one NTPPPH.10,12-14 However, the subcellular distributions and functions of each NTPPPH isozyme may differ in a given cell type, which is believed to reflect in large part substantial differences in the cytosolic tails of each isozyme.12-14
PC-1 potently inhibits hydroxypatite crystal deposition by osteoblasts in vitro.10 Moreover, PC-1 has been demonstrated to serve as a physiological mineralization inhibitor in vivo.21 Specifically, in tiptoe-walking ttw/ttw mice, which have autosomal recessive inheritance of a naturally occurring nonsense truncation mutation in PC-1, hydroxyapatite-containing peri-articular calcifications develop in early life (by 3 weeks of age).21,22 The ttw/ttw mice also develop synovial and articular cartilage, and arterial calcification in early life, and progressive fusion of spinal ligaments and of small and large peripheral joints in early adulthood.21 A nearly identical phenotype to that of ttw/ttw mice was recently observed in PC-1 knockout mice.22 Furthermore, deficient function of a nonenzymatic plasma-membrane PPi channel that supports extracellular PPi levels (termed ANK) has been associated with similar, progressive hyperostosis by early adulthood in ank/ank mice.11
In this study, we characterized a patient with IIAC (and infantile peri-articular calcifications) in which extensive study revealed a PC-1/NTPPPH and extracellular PPi deficiency state. The results presented below indicate that PC-1 deficiency in humans and mice can be associated with similar phenotypic features.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For study of explant-cultured dermal fibroblasts, 1-cm dermal excision biopsies were taken under informed consent by institutionally approved protocol from the medial aspect of the upper forearm of the proband at age 25 months, each unaffected family member, and unrelated normal children (ages 2 to 6 years old) as controls.
Dermal samples were finely minced and then initially cultured for three passages in Chang in situ medium (Irvine Scientific, Irvine, CA), supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. For studies of NTPPPH and PPi metabolism cells were subcultured, from passages 4 to 10, in Medium 106 (Cascade Biologics, Portland, OR) supplemented with 100 U/ml penicillin and 50 µg/ml streptomycin (Omega Scientific, Tarzana, CA), and 1% v/v low serum growth supplement (Cascade Biologics).
PPi was determined and equalized for cell DNA, in samples immediately treated after isolation for 10 minutes at 65°C, conditions verified to heat-inactivate all PPi-synthesizing and PPi-degrading activities.12 For measurements of intracellular PPi, fibroblasts were treated as previously described before the PPi assay,12 which used differential adsorption on activated charcoal of UDP-D-[6-3H]-glucose (Amersham, Chicago, IL) from the PPi-stimulated reaction product 6-phospho-[6-3H]-gluconate.
Heparinized plasmas were obtained from the IIAC proband and family members, and unrelated normal children (ages 18 to 27 months) on several different days, and buffered 1:1 (for storage at -70°C) in 0.2 mol/L Tris, 1.6 mmol/L MgCl2, pH 8.1.12 NTPPPH and alkaline phosphatase-specific activities (per µg plasma or cell protein) were determined by colorimetric assays at alkaline pH using synthetic substrates.12 Protein assays (used to calculate specific activity) were performed using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) using albumin as a standard. For PPi analyses, separate aliquots of plasma samples were heat-treated, as done above for the fibroblast samples.
All assays of constitutive NTPPPH and PPi metabolism, Western blots, and PC-1 mRNA expression, and confocal microscopy studies were done on fibroblasts at passages 4 to 6. Cells from subsequent passages only were used to provide adequate genomic DNA for sequencing purposes.
SMC Culture, Proliferation Assay, and Cell Transfection
Human second passage aortic SMCs from normal donors were obtained from Cascade Biologics and were grown in Medium 231 supplemented with 100 U/ml penicillin and 50 µg/ml streptomycin (Omega Scientific), and proprietary smooth muscle growth supplement (at 5% v/v) from Cascade Biologics. Cells were subcultured every 5 days, and media were resupplemented every 48 hours. For transfection studies, aliquots of 3 x 105 SMCs (fourth passage) were plated in a 35-mm culture dish and allowed to adhere overnight and then transfected with 1 µg of plasmid DNA by a previously described lifofectamine plus protocol.12 Where fibroblasts were transfected, we used only fifth passage cells. We achieved a transfection efficiency of >55% in SMCs and >45% in fibroblasts in these studies.
To measure SMC proliferation, we plated aliquots of 5,000 cells in a 96-well plate and allowed them to adhere for 18 hours. Then the cells were transfected, as described above, incubated for 8 hours, and then carried in fresh medium for 24 hours, at which time the plate was treated with Hoescht H33258 (fluorogenic DNA binding dye) to determine cell numbers (excitation of 355 nm and emission 460 nm). A standard curve was used for each assay to extrapolate the values from fluorescence units to cell numbers.
Western Blotting and Immunoprecipitation Studies
Western blotting for immunodetection of PC-1 and B10/PDNP3 was performed as previously described, using the rabbit anti-PC-1 antibody R1769 specific for the 150 amino acid C-terminal domain of PC-1,23 and a previously described rabbit antiserum to B10/PDNP3 amino acids 193 to 616.12 For detection of autotaxin, we used a rabbit polyclonal antiserum collected after immunization with the KLH-conjugated peptide NH2-TEFLSNYLTNVDDITLVPGTLGR-COOH corresponding to amino acids 370 to 392 of autotaxin and common to full-length autotaxin and the PD-I alpha splice variant.20
For immunoprecipitation of NTPPPH isozymes, 50 µg of plasma protein,
or, where indicated, 50 µg (in protein) of fibroblast lysates (from
5 x 105
cells lysed in 0.1 mol/L Tris,
1.6 mmol/L MgCl2, 1% Triton X-100, pH 8.1) was
precleared with 1:10 volume of protein G-Sepharose beads (Sigma, St.
Louis, MO). Specific antibodies that recognized native human PC-1 (the
monoclonal antibody 3E8, a gift of Dr. J. Goding, Monash Medical
School, Prahran, Australia),12
or the above-described
antisera to autotaxin or B10/PDNP3 (1 µl) were added to each sample,
and then the total volume was brought up to 0.25 ml in 0.1 mol/L Tris,
1.6 mmol/L MgCl2, pH 8.1. After constant rotation
at 4°C overnight, protein G-Sepharose beads (25 µl) were added, and
samples were maintained under constant rotation for an additional 24
hours at 4°C. The beads were then centrifuged, and washed three times
with 0.1 mol/L Tris, 1.6 mmol/L MgCl2, pH 8.1
buffer (without 1% Triton X-100). To allow analysis of bead-associated
proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and Western blotting, beads were treated at 95°C in 0.1 ml of sodium
dodecyl sulfate-containing buffer for 5 minutes.
Confocal Immunofluorescence Microscopy
Poly-D-lysine-coated coverslips were plated with either proband or control explant cultured dermal fibroblasts so as to achieve a cell density of 50 to 75% at 24 hours. In brief, the staining protocol involved fixing cells with 4% paraformaldehyde solution and blocking with 5% bovine serum albumin containing phosphate-buffered saline. PC-1 was detected using the monoclonal antibody 3E8, and B10/PDNP3 was detected using the rabbit polyclonal serum to human B10/PDNP3 cited above, as described.12 Secondary antibodies used were Alexa 488 goat anti-mouse IgG conjugate or Alexa 568 goat anti-rabbit IgG conjugate, both from Molecular Probes (Eugene, OR). Fluorescent images of the cells were visualized by laser confocal microscopy, using a Zeiss Axiovert 100 mol/L laser scanning microscope, as previously described.12
Enzyme-Linked Immunosorbent Assay (ELISA) for Soluble Plasma PC-1
To quantify soluble PC-1 in heparinized plasmas, ELISA plates were coated overnight at 4°C with an affinity-purified polyclonal antibody to PC-1, as previously described.24 All samples were tested in a blinded manner. In brief, after washing with TBST buffer (20 mmol/L Tris, 150 mmol/L NaCl, and 0.05% Tween-20) to remove unbound antibody, wells were blocked with SuperBlock (Pierce Chemical, Rockford, IL). Next, human plasma (10 to 15 µl diluted to a total volume of 100 µl with 50 mmol/L HEPES, pH 7.6, 150 mmol/L NaCl, 0.1% Triton X-100, 1 mmol/L phenylmethyl sulfonyl fluoride, 1 mg/ml bovine serum albumin) was added to wells, and PC-1 in the plasma was allowed to bind overnight at 4°C. After extensive washing, biotinylated anti-PC-1 monoclonal antibody was added to wells for 2 hours. Wells were again washed, and then streptavidin horseradish peroxidase was added for 30 minutes. Next, the signal was enhanced using ELAST (Dupont NEN, Boston, MA), according to the manufacturer’s instructions. The signal was developed using 100 µl of TMB reagent (Kirkegaard-Perry, Gaithersburg, MD), and the reaction was stopped using 100 µl of 1 mol/L H3PO4, with absorbance measured at 451 nm.
Sequencing of PC-1, B10/PDNP3, and Autotaxin
Genomic DNAs from proband, appropriate age-related controls, and family members were PCR-amplified using intron-exon-specific primers for PC-1.25 The entire coding region cDNA sequence of B10/PDNP3, and partial cDNA sequence of autotaxin (limited to domains encompassing the catalytic site and EF hand domain) also were determined using primers that spanned the cDNA regions, generating amplicons of 200 to 350 bp. All polymerase chain reaction products were electrophoretically separated, extracted, and then purified using Qiagen (Valencia, CA) gel extraction columns. Sequencing of all products was performed using dye primer chemistry on an ABI377 automated sequencer. The derived sequences were compared to the published sequences for each NTPPPH isozyme.
Northern Blot Analysis
Explant-cultured dermal fibroblasts were homogenized and total RNA prepared according to the manufacturer’s instructions using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). The RNA samples (20 µg) were resolved on a 1% formaldehyde-agarose gel run at 5 V/cm and then transferred to MagnaGraph nylon membranes (Fisher Scientific, Tustin, CA) by capillary transfer. After transfer, the membrane was UV cross-linked at 254 nm according to the manufacturer’s instructions. Hybridization was performed at 42°C using a PC-1 cDNA probe with specific activity >108 cpm/µg, prepared by using a random primer labeling kit (Life Technologies, Inc.). The PC-1 cDNA probe encompassed the PC-1-specific region encoding the cytosolic tail, and was generated by digesting a pcDNA3 PC-1 construct12 with BamHI and ScaI. Densitometric analysis of autoradiographs was performed as previously described.10
Immunohistochemistry
For immunohistochemical analysis of artery lesion PC-1 and
B10/PDNP3, formalin-fixed and paraffin-embedded tissues from the
proband’s temporal artery biopsy and from unrelated adult
atherosclerotic human carotid endarterectomy samples were sectioned at
3-µm thickness. After deparaffinization and rehydration, appropriate
blocking buffers were applied to the sections. The sections were
incubated with primary antibodies (1:200 of the rabbit antibody to
B10/PDNP3 described above, and 1:100 of the monoclonal anti-human PC-1
antibody 3E8 cited above) for 14 hours at 4°C. After an extensive
wash, the sections were blocked for endogenous peroxidase activity with
Peroxo-Block (Zymed, South San Francisco, CA) and incubated with
secondary species-specific antibody at 5 µg/ml for 1 hour. The
sections were incubated with Vectastain ABC Elite solution (Vector
Laboratories, Burlingame, CA) for 30 minutes and developed with
9-amino-3-ethylene-carbazole (Vector Laboratories) and counterstained
with hematoxylin. We also used anti--actin antibody conjugated with
horseradish peroxidase (DAKO, Carpinteria, CA), and the sections to be
stained for SMC actin were incubated with the antibody for 1 hour,
blocked with Peroxo-Block, and developed with
9-amino-3-ethylene-carbazole. Negative control sections were treated by
using appropriate dilutions of the normal sera of the species in which
the primary antibodies were made.
Gene Linkage Analyses for Matrix GLA Protein (MGP) and PC-1
To test if the disorder was caused by mutation(s) in the matrix GLA protein gene (MGP), the MGP gene was amplified using four sets of primers as described27 and the products were analyzed for single-strand conformation polymorphism. No variant patterns were observed. Subsequently, possible genetic linkage of the disorder to the PC-1 locus on chromosome 6q was tested by analysis of polymorphic variable-number tandem repeats around the region of interest. The analyzed markers were D6S287, D6S1009, and D6S311. The markers were amplified using fluorescence-labeled markers and their length determined using a Perkin-Elmer ABI 310 automated DNA sequence apparatus (Perkin-Elmer, Emeryville, CA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The case history of the proband was recently described in detail.8 In brief, the proband is the male child of a consanguineous marriage (the fathers of each parent were brothers). Because of fetal distress, the proband was delivered preterm by caesarian section in the 36th week of gestation, in January 1997. The mother’s obstetrical history was G9, P4, A2 (spontaneous), with three additional stillborns. None of the surviving family members has developed IIAC.
At 5 days of age, the proband was diagnosed with congestive heart
failure because of dilated cardiomyopathy. By 8 months of age, renal
artery stenoses, aortic calcifications, and wrist and ankle
peri-articular calcifications (Figure 1)
had developed. The proband also started to experience episodes of acute
peri-articular inflammation of the wrists and ankles.
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Blood karyotyping of the proband revealed a normal male karyotype in 50 cells studied. An extensive work-up in the proband ruled out a primary endocrine or metabolic disorder. Initially, we focused on the MGP gene as candidate locus for this disorder, because of the association of arterial calcification with MGP deficiency in mice.28 A screen for mutations using a sensitive single-strand conformation polymorphism assay did not indicate presence of sequence changes in the IIAC proband or in his parents as compared to control individuals (data not shown).
Markedly low urine PPi levels were present in the proband relative to unrelated normals.8 Thus, we pursued investigation of plasma- and explant-cultured fibroblast PPi, and of PPi-generating NTPPPH isozymes.
Deficiencies of PC-1, NTPPPH Activity, and Extracellular PPi Levels in the Proband
We first assessed levels of soluble plasma PC-1 by ELISA (in a
blinded manner for the laboratory performing the ELISA). Because there
is no normative data for PC-1 levels in plasma, sampling was generally
done at more than one time point. Normal control children and
unaffected family members had PC-1 plasma levels in the range of 10 to
30 ng/ml (Figure 2A)
. In contrast, the
proband consistently demonstrated trace PC-1 levels (<1 ng/ml) in
plasma.
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. We also observed a consistent,
relative depression total plasma NTPPPH-specific activity levels in the
proband (a mean of <5 U/µg protein) (Figure 2C)
, similar overall to
the results for plasma PPi in the proband relative to unrelated
controls. Plasma NTPPPH-specific activity levels also remained
consistently lower (again a mean of <5 U/µg protein in three
separate determinations) in the proband than in the unaffected family
members (Figure 2C)
. Results in all of these experiments were the same
whether PPi or NTPPPH were measured as levels per µg protein, as
shown, or per µl volume (not shown).
Some variability in plasma-specific activity of total NTPPPH was
apparent in the proband’s mother (FE) and the brother (OE), whose
NTPPPH-specific activity levels (6 and 8 U/µg protein each on
separate occasions) approached to the range of plasma NTPPPH activity
(4 to 5 U/µg protein) observed in the proband (Figure 2C)
. Thus,
we further studied NTPPPH-specific activity (and intracellular and
extracellular PPi) in explant-cultured dermal fibroblasts (Figure 3)
. We observed markedly lower
NTPPPH-specific activity, and intracellular and extracellular PPi
levels, in cells from the proband relative to all of the unaffected
family members, and to two additional unrelated children controls.
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Confocal immunofluorescence microscopy studies demonstrated at
most trace levels of PC-1 in proband explant-cultured fibroblasts in
comparison to several controls (Figure 4)
. Furthermore, in proband fibroblasts,
PC-1 was sparse in comparison to B10/PDNP3, as opposed to the
concomitant abundance of both isozymes in control fibroblasts (Figure 4)
.
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130 kd under
reducing conditions) (Figure 5)
. We
similarly recovered autotaxin/PD-1 and B10/PDNP3 of normal size from
proband plasma, and fibroblasts, respectively (not shown). We also used
immunoprecipitation to screen for the presence of NTPPPH enzymatic
activity associated with individual NTPPPH isozymes. Using polyclonal
antibodies specific for autotaxin/PD1, as described in Materials and
Methods, we consistently immunoprecipitated approximately half of the
NTPPPH in normal plasma and >80% of plasma NTPPPH activity in the
proband (not shown). Polyclonal antibodies specific for B10/PDNP3, and
to a lesser degree monoclonal antibody specific for PC-1 each were able
to immunoprecipitate small but significant levels of functional NTPPPH
activity in fibroblast lysates from the proband (not shown).
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. The entire
coding regions of PC-1 and B10/PDNP3 were sequenced, as were fragments
of the autotaxin/PD1 coding region encompassing the catalytic site
and EF hand domain, and we did not find significant coding region
sequence mutations in the proband (not shown). Thus, the proband
cells had deficient PC-1 expression but were capable of
expressing normal PC-1 and of generating an increase in extracellular
PPi in response to forced expression of PC-1.
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.
Specifically, three haplotypes segregated in the nuclear family.
Neither the proband nor his unaffected siblings were homozygous by
descent. The proband had the same haplotype as his mother.
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Studies to this point demonstrated deficient PC-1/NTPPPH
expression in the proband. Because IIAC was the dominant clinical
feature in the proband, we immunohistochemically analyzed the temporal
artery biopsy from the proband. We observed marked expansion of SMCs
bordering the internal elastic lamina, abundant expression of the
NTPPPH isozyme B10/PDNP3 by the expanded population of SMCs, but sparse
PC-1/NTPPPH expression by the same cells (Figure 8)
. In contrast, SMCs from human carotid
atherosclerotic lesions of two adults unrelated to the proband
demonstrated readily detectable PC-1 expression (and little B10/PDNP3
expression) in the media in association with SMCs (Figure 8)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and B10/PDNP3,
have been observed to have distinct subcellular distributions and
functions when compared to PC-1 in hepatocytes, chondrocytes, and
osteoblasts.12-14
For example, the autotaxin splice
variant of the autotaxin/PD1 gene, is predominantly
secreted.18,19
Furthermore, B10/PDNP3, unlike PC-1, is not
enriched in the chondrocyte plasma membrane and although B10/PDNP3
regulates intracellular PPi concentrations it does not seem to
significantly regulate extracellular PPi in
chondrocytes.12
In addition, PC-1 but not B10/PDNP3
suppresses osteoblastic matrix vesicle-mediated mineralization in
vitro.29
The association of deficient PC-1 expression with human IIAC in the
case studies occurred despite the findings that autotaxin-associated
NTPPPH activity appeared to be abundant in proband plasma. Furthermore,
B10/PDNP3, which had a normal cDNA sequence and was of normal
polypeptide size in the proband, was abundantly expressed relative to
PC-1 in both proband fibroblasts and the expanded population of SMCs in
the arterial lesions of the proband. Thus, the physiological function
of PC-1 as a mineralization inhibitor seems to be unique and not
readily compensated for by NTPPPH activities of B10/PDNP3 or
autotaxin/PD-1.
We observed that PC-1, which was expressed by normal cultured aortic SMCs and SMCs in human atherosclerotic lesions, functioned at least in part to increase the extracellular levels of the mineralization inhibitor PPi in vitro. In this study, PC-1 expression was barely detectable in a markedly expanded population of SMCs in the proband temporal artery lesions. Taken together, the results suggested that a loss of PC-1-related homeostatic functions in the wall of the temporal artery might be central to IIAC pathogenesis at a local level in the artery wall. However, interpretation of these data are inherently limited because we did not carry out comparisons (to the proband) of relative PC-1 and B10/PDNP3 expression in temporal arteries of other family members and of unrelated children and adults.
Because transfection of wild-type PC-1 in proband cells induced a significant increase in both NTPPPH activity and extracellular PPi (data not shown), a primary PC-1 translational or posttranslational processing defect seemed unlikely in the proband. Defective function of the membrane PPi channel ANK is associated with low extracellular PPi and elevated intracellular PPi in fibroblasts.11 However, both intracellular PPi and extracellular PPi were low in proband fibroblasts, which was consistent with a primary defect in PC-1/NTPPPH activity.12 Therefore, ANK expression and function were not specifically explored in the proband.
In the temporal artery of the IIAC proband, we observed marked expansion of the SMC population. Ingestion by SMCs of hydroxyapatite, deposited in the arterial wall in IIAC associated with deficient PPi production, could by itself stimulate SMC proliferation, analogous to mitogenic effects of ingested hydroxyapatite in fibroblasts.30 However, we speculate that PC-1 might function to prevent arterial calcification by not only modulating PPi generation but also by other effects on SMC differentiation and/or regulation of SMC matrix proteoglycan synthesis via PC-1 nucleotide pyrophosphatase activity on the sulfation donor phosphoadenosine phosphosulfate.13,14 Although transfection of PC-1 did not affect proliferation of normal cultured SMCs in vitro, it remains to be determined if marked PC-1 deficiency affects SMC proliferation in vivo.
The PC-1-deficient IIAC proband studied here in early childhood had peri-articular calcifications, which also develop in early childhood in PC-1-deficient mice.21-23 Certain phenotypic features, including osteophytes, spinal ligament hyperostosis, and articular cartilage calcifications, develop at later points in life in PC-1-deficient mice.21-23 More prolonged observation of the proband will be of interest, but the future phenotype of the proband will likely be affected by his treatment with bisphosphonates. It is possible that PC-1 may not play as broad a regulatory role for calcification in humans, as compared to mice, but it may not be possible to determine this by future study of the proband.
Immunoreactive soluble PC-1 was dramatically reduced in proband plasma and full-length PC-1, although detectable by immunoprecipitation, also was relatively sparse in cultured fibroblasts from the proband. Interestingly, the proband was heterozygous for PC-1. Given the heterozygosity of the IIAC proband for PC-1, it was not surprising to observe that the IIAC proband dermal fibroblasts expressed at least some PC-1 polypeptide (of normal size). In addition, proband PC-1 mRNA was normal in size.
The PC-1 expression defect of the proband seemed to be more profound for circulating PC-1 and for PC-1 expression in the diseased temporal artery wall than in the proband fibroblasts cultured under proliferative conditions in serum-containing media. Further studies to determine the molecular genetic basis for the relative but not absolute PC-1 deficiency in the proband will be of interest. Although the combination of the proband’s PC-1 haplotypes was shared with his mother, it remains possible that their PC-1 genotype will be found to demonstrate distinctions. Genetic linkage studies will be limited by the fact that only one surviving member of the kindred was affected. We were unable to identify abnormalities in the coding region sequence of PC-1 in the proband. However, it is recognized that a variety of types of mutations can be difficult to identify, particularly in heterozygous patients.31 Conversion approaches31 to identifying a possible refractory PC-1 mutation in the proband will be of interest to apply in the future. Included among anomalies that could be envisaged in the proband are polymorphism(s) or mutation(s) of the PC-1 promoter that might result in decreased transcription activation in response to PC-1 expression-inducing growth factors such as transforming growth factor-{beta}, a-fibroblast growth factor and b-fibroblast growth factor in developing arteries and peri-articular tissues.32 Another possibility would be mutation of a receptor or transcription factor that functions to drive (or suppress) PC-1 expression, particularly in vascular SMCs.
In mice, PC-1 is one of several genes for which homologous recombination-induced deficiency has been noted to be associated with spontaneous arterial calcification.1,2 However, mice deficient in the osteoclast differentiation inhibitory factor osteoprotegerin develop arterial calcification but not peri-articular calcification in early life.33 Although mice deficient in MGP develop spontaneous arterial calcification they also manifest pathological cartilage calcification.28 Furthermore, deficient MGP expression in humans (Keutel’s syndrome) has thus far not been associated with arterial calcification.34 Screening for MGP mutations was negative in the affected kindred. Because we did not sequence the entire MGP or osteoprotegerin genes in our family, or screen for their expression in arterial lesions, we did not exclude a primary or secondary contribution of mutations or abnormalities in expression of either of these genes to IIAC in the proband. However, given the phenotype of combined IIAC and peri-articular calcification in the proband and the marked abnormalities in NTPPPH activity and PPi metabolism, the possibility that either MGP deficiency or osteoprotegerin deficiency were the driving force for the calcification disorder seems unlikely.
In conclusion, the results of this study indicate the utility of screening for plasma and tissue PC-1, NTPPPH, and PPi levels in evaluating IIAC, particularly when associated with peri-articular calcification. PC-1 may ultimately serve as a therapeutic target in certain cases of IIAC.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Department of Veterans Affairs, National Institutes of Health (P01AGO7996, HL61731, DK52999), the Arthritis Foundation, the Swiss National Science Foundation (grant 31-57272.99) and the Ministry of Education, Science, Sports, and Culture of Japan.
F.R. and S.V. contributed equally to this work.
Accepted for publication October 20, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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X. Li, H.-Y. Yang, and C. M. Giachelli Role of the Sodium-Dependent Phosphate Cotransporter, Pit-1, in Vascular Smooth Muscle Cell Calcification Circ. Res., April 14, 2006; 98(7): 905 - 912. [Abstract] [Full Text] [PDF]
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 K. A. Lomashvili, W. Khawandi, and W. C. O'Neill Reduced Plasma Pyrophosphate Levels in Hemodialysis Patients J. Am. Soc. Nephrol., August 1, 2005; 16(8): 2495 - 2500. [Abstract] [Full Text] [PDF]
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 H. C. Anderson, D. Harmey, N. P. Camacho, R. Garimella, J. B. Sipe, S. Tague, X. Bi, K. Johnson, R. Terkeltaub, and J. L. Millan Sustained Osteomalacia of Long Bones Despite Major Improvement in Other Hypophosphatasia-Related Mineral Deficits in Tissue Nonspecific Alkaline Phosphatase/Nucleotide Pyrophosphatase Phosphodiesterase 1 Double-Deficient Mice Am. J. Pathol., June 1, 2005; 166(6): 1711 - 1720. [Abstract] [Full Text] [PDF]
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C. M. Giachelli, M. Y. Speer, X. Li, R. M. Rajachar, and H. Yang Regulation of Vascular Calcification: Roles of Phosphate and Osteopontin Circ. Res., April 15, 2005; 96(7): 717 - 722. [Abstract] [Full Text] [PDF]
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D. A. Towler Inorganic Pyrophosphate: A Paracrine Regulator of Vascular Calcification and Smooth Muscle Phenotype Arterioscler. Thromb. Vasc. Biol., April 1, 2005; 25(4): 651 - 654. [Full Text] [PDF]
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K. Johnson, M. Polewski, D. van Etten, and R. Terkeltaub Chondrogenesis Mediated by PPi Depletion Promotes Spontaneous Aortic Calcification in NPP1-/- Mice Arterioscler. Thromb. Vasc. Biol., April 1, 2005; 25(4): 686 - 691. [Abstract] [Full Text] [PDF]
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 H. Dong, B. A. Maddux, J. Altomonte, M. Meseck, D. Accili, R. Terkeltaub, K. Johnson, J. F. Youngren, and I. D. Goldfine Increased Hepatic Levels of the Insulin Receptor Inhibitor, PC-1/NPP1, Induce Insulin Resistance and Glucose Intolerance Diabetes, February 1, 2005; 54(2): 367 - 372. [Abstract] [Full Text] [PDF]
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K. A. Lomashvili, S. Cobbs, R. A. Hennigar, K. I. Hardcastle, and W. C. O'Neill Phosphate-Induced Vascular Calcification: Role of Pyrophosphate and Osteopontin J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1392 - 1401. [Abstract] [Full Text] [PDF]
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 S. M. Vaingankar, T. A. Fitzpatrick, K. Johnson, J. W. Goding, M. Mauri |
