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Type V OI primary osteoblasts display increased
mineralization despite decreased COL1A1 expression
Adi Reich
1
, Alison S. Bae
1
, Aileen M. Barnes
1
, Wayne A. Cabral
1
,
Aleksander Hinek
2
, Jennifer Stimec
3
, Suvimol C. Hill
4
, David Chitayat
5,6
, and
Joan C. Marini
1
1Bone and Extracellular Matrix Branch, NICHD, NIH, Bethesda, MD, USA;
2
Physiology and Experimental
Medicine Program, Heart Center, Hospital for Sick Children, University of Toronto, ON, Canada;
3
Division of Diagnostic Imaging, Department of Pediatrics, Hospital for Sick Children, University of
Toronto, Toronto, ON, Canada;
4
Diagnostic Radiology Department, NIH Clinical Center, NIH, Bethesda,
MD, USA;
5
The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and
Gynecology, Mt Sinai Hospital, University of Toronto, Toronto, ON, Canada;
6
Division of Clinical and
Metabolic Genetics, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto,
ON, Canada
Context: Patients with type V osteogenesis imperfecta (OI) are heterozygous for a dominant IFITM5
c.-14C!T mutation, which adds 5 residues to the N-terminus of BRIL, a transmembrane protein
expressed in osteoblasts. Type V OI skeletal findings include hyperplastic callus formation, ossifi-
cation of the forearm interosseous membrane and dense metaphyseal bands.
Objective: The objective of this study was to examine the role of osteoblasts in the active miner-
alization traits of type V OI, and the effect of the IFITM5 mutation on type I collagen.
Methods: We identified 8 patients with the IFITM5 c.-14C!T mutation. Cultured osteoblasts from
type V OI patients were used to study osteoblast differentiation and mineralization.
Results: We verified expression and stability of mutant IFITM5 transcripts. In differentiated type V
OI primary osteoblasts in culture, IFITM5 expression and BRIL protein level is comparable to control,
Both early and late markers of osteoblast differentiation are increased in type V OI osteoblasts.
Mineralization, assayed by alizarin red staining, was increased in type V OI osteoblasts compared
to control. However, type V OI osteoblasts have significantly decreased COL1A1 transcripts in mid
to late differentiation. Type I collagen protein is concomitantly decreased, with decreased cross-
linked collagen in matrix, and altered appearance of fibrils deposited in culture.
Conclusions: This study demonstrates that type V OI mineralization has a gain-of-function mech-
anism at the osteoblast level, which likely underlies the overactive tissue mineralization seen in
patients. Decreased type I collagen expression, secretion and matrix incorporation establish type
V OI as a collagen-related defect.
Osteogenesis imperfecta (OI) is a genetically heteroge-
neous heritable connective tissue disorder charac-
terized by intrinsic bone fragility, resulting in frequent
fractures and deformities of the long bones and spine (1).
The phenotypic severity of OI ranges from perinatal lethal
to subtle fracture susceptibility and generalized osteope-
nia (2). Most cases of osteogenesis imperfecta are caused
by autosomal dominant mutations in the genes encoding
type I collagen, COL1A1 or COL1A2 (3). Autosomal re-
cessive osteogenesis imperfecta, with lethal to moderate
phenotypes, is caused by defects in genes whose products
interact with type I collagen for folding, post-translation
modification or processing (4). Most recessive cases have
null mutations in genes whose proteins are involved in
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received July 31, 2014. Accepted October 31, 2014.
Abbreviations:
ORIGINAL ARTICLE
Endocrine Research
doi: 10.1210/jc.2014-3082 J Clin Endocrinol Metab jcem.endojournals.org 1
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collagen prolyl 3-hydroxylation (CRTAP,LEPRE1 and
PPIB) (5–7) or proper helical folding (FKBP10 and SER-
PINH1) (8–11). Additional genes causing recessive OI
include SERPINF1, which encodes pigment epithelium-
derived factor (PEDF) (12, 13), collagen C-propeptide
cleavage enzyme (BMP1) (14), and WNT1 (15, 16).
Type V OI is the only dominantly inherited form of OI
not caused by mutations in COL1A1 or COL1A2 (4). It
has distinctive clinical and radiographic features, includ-
ing variable occurrence of hyperplastic callus, calcifica-
tion of the forearm interosseous membrane, radial-head
dislocation, and a subphyseal metaphyseal radiodense
band (17, 18). The histomorphometry of type V OI is also
distinct, with a mesh-like lamellation pattern (17). Recent
reports have shown that type V OI is caused by a single
recurrent heterozygous mutation in Interferon-Induced
Transmembrane Protein 5 (IFITM5), which encodes
Bone-restricted (IFITM)-like protein (BRIL), a protein in-
volved in mineralization and expressed in the skeleton (19,
20). The type V OI mutation is a C!T transition at posi-
tion –14 of the 5"untranslated region (UTR) of IFITM5
(c.-14C!T) (21, 22). This mutation generates a new start
codon, adding five amino acids to the N-terminus of the
protein. However, the mutant protein maintains the same
intramembrane topology and palmitoylation as normal
BRIL (23), and has been speculated to have a gain-of-
function mechanism. The understanding that the same
mutation is responsible for all cases of type V OI then led
to recognition of phenotypic variability (24). We describe
here 8 patients with type V OI from 5 different families,
who display phenotypic variability.
The mechanism of type V OI is not understood at the
cellular level, in this case, the bone forming osteoblasts.
Using primary cultured osteoblasts from type V OI pa-
tients, we verified the expression of mutant IFITM5 tran-
script and protein. We utilized these primary mutant os-
teoblasts to demonstrate that the presence of the IFITM5
mutation increases multiple markers of osteoblast differ-
entiation. Type V OI osteoblasts also display increased
mineral deposition in culture, demonstrating that a gain-
of-function mechanism at the cellular level underlies the
active mineralization traits in type V OI patients. Despite
increased osteoblast developmental markers, COL1A1
expression, secretion and deposition in matrix by type V
OI osteoblasts is significantly decreased, which establishes
type V OI as a collagen-related dysplasia.
Materials and Methods
Patients and Cells
Patients 2, 3, 5, and 8 are patients at the NIH Clinical Center,
whose samples were collected under an IRB approved protocol.
Osteoblasts were outgrowths from bone chips collected as sur-
gical discard during medically indicated orthopedic procedures.
patient 7 is followed at the Genetic Clinic at Mt Sinai Hospital,
Toronto, Ontario, Canada; samples were collected with parental
consent. Detailed case reports on the patients are presented in
Supplemental Material. Control fibroblasts were ATCC line
2127, which is well-validated for collagen biochemistry, while
control osteoblasts were obtained with parental consent from
surgical discard bone chips of an unaffected child during an elec-
tive orthopedic procedure. These control cells have been used
extensively in our lab and validated for experiments involving
osteoblast development and collagen biochemistry.
Mutation identification and verification
We screened genomic DNA (gDNA) of dermal fibroblasts,
leukocytes, or both, from the control, patients, parents or unaf-
fected siblings. Sequencing of complementary DNA (cDNA) and
gDNA from the patients revealed no mutations in COL1A1,
COL1A2,CRTAP,LEPRE1,PPIB,SERPINH1,FKBP10 or
SERPINF1. All patient skin and bone biopsies were obtained
with informed consent under a protocol approved by the
NICHD IRB.
The two exons and flanking intronic sequences of IFITM5
gDNA from leukocytes of control, patients, sibling and parents
were amplified by PCR, as previously described (25) and se-
quenced. Patient and control IFITM5 cDNA from fibroblasts
was also sequenced and the mutation was confirmed by BsmAI
restriction enzyme digestion.
Cell culture
Dermal fibroblast (FB) cultures were established from skin
punch biopsies. FB were grown in Dulbecco’s Modified Eagle
Medium (DMEM) (Life Technologies, Grand Island, NY) con-
taining 10% fetal bovine serum (FBS), 100 U/ml penicillin and
100
!
g/ml streptomycin. Osteoblast (OB) primary cultures were
established from surgical bone chips (26) of normal control and
OI patients who had not received bisphosphonates. OB were
cultured in MEM alpha (Life Technologies) supplemented with
10% FBS, penicillin and streptomycin at 37°C and 8% CO
2
.
Osteoblast cultures were grown to confluence, then treated with
osteoblast differentiation medium (containing 25
!
g/ml L-ascor-
bic acid, 10
-8
M Dexamethasone and 2.5 mM 2-glycerophos-
phate, with or without 100 ng/ml human recombinant BMP2
(the generous gift of Wyeth, Dallas, TX)).
RT-PCR
Total RNA was extracted from control and patient primary
osteoblast and fibroblast cultures using TriReagent (Molecular
Research Center, Cincinnati, OH). cDNA was reverse tran-
scribed from 5
!
g RNA using a High-Capacity cDNA Archive
Kit or MuLV reverse transcriptase and Oligo d(T)
16
(Life Tech-
nologies, Grand Island, NY). Transcript levels of IFITM5
(Hs00942485 g1), RUNX2 (Hs00231692 m1), COL1A1
(Hs00164004 m1), ALPL (Hs01029144 m1), BGLAP
(Hs01587814 g1), SPP1 (Hs00959010 m1), SERPINF1
(Hs01106934 m1), and IBSP (Hs00173720 m1) (Life Tech-
nologies) were determined with TaqMan Gene Expression As-
says on a 7500 Fast Real-Time PCR System (Applied Biosys-
tems). Relative expression of each gene of interest was performed
in triplicate and normalized to expression of GAPDH
(Hs99999905 m1). Expression levels were compared with age-
2Increased mineralization of type V OI Osteoblasts J Clin Endocrinol Metab
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matched control osteoblasts (Day 1 or 5). Expression of IFITM5
in osteoblasts and fibroblasts by PCR (as shown in Figure S1C)
was demonstrated using primers in exon 1 (F: 5!-TTGATCTG-
GTCGGTGTTCAG-3!) and exon 2 (5!-GTCAGTCATAGTC-
CGCGTCA-3!) as previously described (21) and generated the
expected 292 bp product.
Western Blot
Cell lysates were collected in RIPA buffer supplemented with
a protease inhibitor cocktail (
!
–Aldrich, St. Louis, MO). Osteo-
blast conditioned media was collected after incubation for 24
hours in serum-free media and supplemented with protease in-
hibitors. Media was concentrated using centrifugal filters (EMD
Millipore, Billerica, MA) and normalized to cell counts for equal
loading.
Proteins were separated on precast 4%–15% Ready Gels
(Bio-Rad, Hercules, CA), transferred to 0.2
"
m nitrocellulose
membranes, and blocked with 5% bovine serum albumin (BSA)
plus 1"casein in PBS before probing with antibody overnight in
2.5% BSA and 0.5"casein. Antibodies used were as follows:
IFITM5 (Abcam, Cambridge, MA), COL1A1 LF-68 (the gener-
ous gift of Dr. Larry W. Fisher, NIH), and actin (
!
–Aldrich).
Blots were washed, incubated with secondary IR-conjugated an-
tibodies for 1 hour, washed, and visualized on a LI-COR Odys-
sey infrared imager (LI-COR, Lincoln, NE).
Mineralization Assay
Osteoblast cultures were grown to confluence in 12-well cul-
ture dishes and stimulated with mineralization medium contain-
ing 25
"
g/ml L-ascorbic acid, 10
-8
M Dexamethasone and 2.5
mM 2-glycerophosphate. Mineralization of the nodules was de-
termined by Alizarin Red-S staining. Cells were washed with PBS
and fixed in 4% paraformaldehyde in PBS for 1 hour, washed
with water and incubated in 1% Alizarin Red-S in 2% ethanol,
followed by a final water wash. Alizarin Red was solubilized with
0.5 ml of 0.5N HCl/5% SDS and absorbance was measured with
spectrophotometer at 405nm (27).
Steady-state Collagen
Control and patient fibroblasts or osteoblasts were grown to
confluence in 6-well culture dishes. Steady-state type I collagen
analysis was conducted as previously described (28). Cells were
incubated with 437.5
"
Ci/ml L-[2,3,4,5-
3
H] proline for 16–18
hours, prior to collection and ammonium sulfate precipitation,
then pepsin-digested and analyzed by 6% SDS-urea-PAGE.
Matrix Deposition
Postconfluent osteoblasts were stimulated with 100
"
g/ml
ascorbic acid three times a week for 14 days then labeled for 24
hours with 400
"
Ci/ml L-[2,3,4,5-
3
H] proline. The media and
extracellular matrix were harvested as described previously (29).
The
3
H-proline-labeled collagens were sequentially extracted
from the matrix fraction with neutral salt, acetic acid and pepsin,
then electrophoresed on 6% SDS-urea-PAGE. Collagen content
of each fraction was measured by densitometry and normalized
to total sample volume.
Immunofluorescence Microscopy
Fibroblasts and osteoblasts were grown to confluence on
chamber slides, then treated with 200
"
M ascorbic acid every
day for 5 days and fixed in 4% paraformaldehyde. Staining of
extracellular matrix was performed essentially as described (30).
The matrix was blocked in 1% BSA in PBS plus 0.02% Tween-
20, washed with PBS and incubated with primary antibody (LF-
68,
#
1(I) C-telopeptide) in 1% BSA/PBS. After washing, the ma-
trix was incubated with secondary antibody, then washed slides
were mounted with DAPI (Vector Laboratories, Burlingame,
CA), and imaged using a Zeiss LSM 510 Inverted Meta micro-
scope and LSM510 software.
Statistical analysis
The data were analyzed by Excel using repeated-measures T
test. Values are presented as mean #SD unless noted. Signifi-
cance was achieved at P$.05.
Results
Identification of Type V OI patients and
phenotypic variability
We identified 8 affected individuals from 5 families
who are heterozygous for the IFITM5 c.-14C$T mutation
found in all individuals with type V OI. Seven patients
were classified clinically as type V OI prior to sequencing,
while patient 8, an adult with progressive deforming OI
not previously classified as type V OI, was found to have
the same mutation. For each patient, the mutation was
confirmed by BsmAI restriction digest; the mutation elim-
inates a BsmAI restriction site (Figure S1C). Sequencing of
patients and family members, as well as patients’ Case
Reports are detailed in the Supplemental material (Figure
S1A, B; Table 1, Table S1).
Type V OI is understood to have variability of clinical
manifestations and of the timing of their appearance (17,
18). Patients 2, 3 and 5 (Figure S3–5) have characteristic
bone histology and radiographic findings. Patients 1, 4, 6
Table 1. Patients Characteristics
Patient Age Sex
Hypertrophic
callus
Radial head
dislocation
Forearm interosseous
membrane calcification Sclera Scoliosis
Age at first
fracture Height (50th centile for age)
Age of diagnosis
of OI type V
1Deceased at 44 yr M %Unknown Unknown Normal Yes Unknown 7-year-old Post mortem
227 yr F %%% Blue Mild in utero, 7 months 6.5-year-old 11-years-old
326 yr M %%% Normal Yes birth 12.5-year-old 10-years-old
450 yr F %Unknown Unknown Blue Yes Unknown 3.5-year-old 33-years-old
522 yr M %%% Blue Mild in utero, birth 2.5-year-old 6-years-old
615 yr M %Unknown Unknown Blue No 1 month 4.5 months at age 30 months before 3 months
75 yr M %%% Blue Yes in utero At CA 3.8 HA 19 months 5-years-old
8Deceased at 73 yr F - %Unknown Normal Yes 18 months 3-year-old Post mortem
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and 7 (Figure S6) have typical radiographic findings, but
bone histology was not examined. The patients in all five
families developed at least one of the classic characteristics
of type V OI. Patients 1, 2, 3, 5, 6 and 7 developed hy-
pertrophic callus. Radial head dislocation was noted in
patients 2, 3, 5, 7 and 8, and ossification of the forearm
interrosseous membrane was documented in patients 2, 5
and 7. Although the initial clinical description of type V OI
reported white sclerae (17, 18), patients 2, 5, 6 and 7 have
blue sclerae (Table 1, Table S1).
IFITM5 expression in osteoblasts and fibroblasts
IFITM5 transcripts were detected by RT-PCR in both
fibroblasts and osteoblasts from normal controls, with the
expected higher expression in osteoblasts than fibroblasts
(Figure S1D). IFITM5 transcripts were also detected in
cultured cells from patients 3 and 7 (Figure S1D). Sanger
sequencing was used to confirm expression of mutant IF-
ITM5 transcripts containing the 5!-terminal addition.
These transcripts could be detected in total RNA from
untreated cells, indicating they have normal stability (Fig-
ure S1E).
In osteoblasts from type V OI patients and control,
IFITM5 expression was barely detectable by real-time RT-
PCR throughout differentiation
with osteogenic media, but addition
of BMP2 significantly and compara-
bly increased IFITM5 expression in
both control and type V osteoblasts
(Figure 1A, left). In differentiated os-
teoblasts, BRIL protein level was
generally comparable in type V OI
and control cells (Figure 1A, right).
IFITM5 mutation affects
osteoblast markers and
mineralization
Multiple markers of osteoblast
differentiation have significantly in-
creased expression in type V OI os-
teoblasts from patient 3 during a dif-
ferentiation timecourse (Figure 2A).
While the preosteoblast marker
RUNX2 had equivalent expression
in mutant and control osteoblasts,
early osteoblast marker alkaline
phosphatase (ALPL) expression was
3 to 6-fold greater than control
throughout differentiation, and the
mid-differentiation marker bone sia-
loprotein (IBSP) (31) peaked on Day
12 at "3-fold greater than control.
Osteocalcin (BGLAP/OCN) and os-
teopontin (SPP1/OPN) are markers of late osteoblast mat-
uration (31). Their peak expression in type V osteoblasts
is about 4-fold greater than control and came late in the
timecourse (Day 15), with BGLAP/OCN expression ris-
ing over time while SPP1/OPN had a late sharp peak. The
addition of BMP2 to maintain IFITM5 expression during
osteoblast differentiation increased transcript levels of dif-
ferentiation markers but maintained their relative level in
type V and control osteoblasts (Figure 2B). Type V OI
osteoblast ALPL, BGLAP and SERPINF1 levels re-
mained greater than control on Day 10 in BMP2 treated
cells.
This acceleration of osteoblast markers was coordi-
nated with increased osteoblast mineralization. Mineral-
ization of differentiating osteoblasts was measured by aliz-
arin red staining. Type V OI osteoblasts displayed a more
brisk mineralization response than did control, increasing
earlier in differentiation and remaining elevated through
Day 15 (Figure 1B).
IFITM5 mutation affects type I collagen expression,
secretion and matrix incorporation
Expression of COL1A1 in type V OI osteoblasts from
2 typical patients (patients 3 and 5), with hyperplastic
Figure 1. IFITM5 expression and osteoblast mineralization A) Left, Relative IFITM5 expression in
control (C) and patient 3 (OI V) osteoblasts (OB) stimulated with osteogenic media for 10 days
with or without addition of BMP2. patient 3 shows normal IFITM5 expression levels by qPCR (n #
3). Right, Representative BRIL western blot from OB stimulated with osteogenic media showing
protein stability in patient (n #3). B) In vitro mineralization assays of control (C) and patient 3
(OI V) OB assessed by alizarin red staining shows increased mineralization of OI V OB at earlier
time points than control (n #3). *P$.05
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callus, calcification of the forearm interosseous mem-
brane, and radial-head dislocation, peaked at Day 5, and
was significantly (2–3 fold) lower than control during mid
to late differentiation (Figure 2A; Figure S2A). The relative
decrease in COL1A1 expression on day 10 was partially
rescued by addition of BMP2 but was still significantly
lower than control (Figure 2B). Electrophoretic migration
of steady-state collagen synthesized by osteoblasts of a
type V OI patient (patient 3) with a typical phenotype
(Figure 3C) is normal. Fibroblast collagen from several,
but not all, type V patients displayed reproducible migra-
tion anomalies which did not correlate with phenotype
(Figure S2B).
Patient collagen protein secreted during osteoblast dif-
ferentiation reflected transcript levels and was reduced
compared to control (Figure 3A). Secreted collagen pro-
tein was quantified as total secreted
!
1(I) chain, by com-
bining measurements of all detected
!
1(I) forms at each
timepoint. Interestingly, collagen from type V osteoblasts
appears to be processed more rapidly in the pericellular
space than does collagen from control osteoblasts, with
relatively more proband mature
!
1(I), or partially pro-
cessed pC
!
1(I), than pro
!
1(I). We do not currently un-
derstand the mechanism of this difference.
Type V OI cultured osteoblasts deposit substantially
less (!4-fold) collagen into matrix fractions containing
crosslinks, with notable reduction in crosslinked
"
-forms
(Figure 3B). Matrix deposited by Type V OI fibroblasts
(Figure 3D, left) and osteoblasts (Figure 3D, right) in long-
term culture was stained for collagen. Type V OI matrix
had fewer well-delineated fibrils and more amorphous
ground material. Collagen fibril packing in patient matrix
was patchy compared to control, with parallel fibril bun-
dles rather than the network appearance of control.
Figure 2. Expression of osteoblast differentiation markers A) Relative expression of Runt-related transcription factor 2 (RUNX2), Alkaline
Phosphatase (ALPL), Bone sialoprotein (IBSP), type I collagen
!
1(COL1A1), Osteopontin (SPP1/OPN) and Osteocalcin (BGLAP/OCN) in control (C)
and patient 3 (OI V) OB stimulated with osteogenic media for up to 15 days. patient 3 shows increased expression of ALPL, IBSP, OPN, and OCN
genes throughout the timecourse (n "3). *P#.05 B) Relative expression of RUNX2,ALPL,COL1A1,SERPINF1 and OCN, in control (C) and
patient 3 (OI V) OB on Day 10 of stimulation with osteogenic media with or without addition of BMP2. patient 3 shows significantly increased
expression of ALPL,SERPINF1 and OCN $BMP2, however, COL1A1 expression $BMP2 is significantly decreased.
doi: 10.1210/jc.2014-3082 jcem.endojournals.org 5
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Discussion
Type V OI is an autosomal dominant form of OI (1, 17).
It is the only rare type of OI which shares the inheritance
pattern of classical OI types I-IV, which are caused by
mutations in the genes that code for type I collagen,
COL1A1 and COL1A2 (1, 2, 4). Type V OI was first
described with clinical criteria over a decade ago, based on
distinctive radiographic and bone histology features (17).
In 2012, two teams of investigators reported that a unique
heterozygous mutation in IFITM5 (21, 22), which en-
codes the protein BRIL, was responsible for all cases of
type V OI (24, 32, 33). BRIL is a transmembrane protein
expressed predominantly in osteoblasts (19, 20), where it
has a Type II orientation in the plasma membrane with the
N-terminal end in the cytoplasm (23). BRIL is also teth-
ered to the membrane by S-palmitoylation modifications
at Cys52 and Cys53, which insert into the cytoplasmic side
of the membrane (23, 34). In the osteoblast membrane, the
association of BRIL with FKBP11 is promoted by S-pal-
mitoylation of the cysteine residues and leads to a larger
complex containing CD81 and CD9 (34).
Two distinct heterozygous IFITM5 mutations have
been reported in OI patients: 1) the c.-14C!T mutation at
the 5"-end of IFITM5 which generates a new start codon,
adding 5 residues to the N-terminal end of BRIL but leav-
ing membrane insertion intact (21, 22): 2) a missense mu-
tation c.119C!T resulting in an p.S40L substitution (25,
35, 36) that interferes with palmitoylation and membrane
insertion (23). All cases of type V OI are caused by the
5"-end mutation, while the p.S40L substitution causes an
atypical progressive deforming OI with the typical bone
histology of type VI OI (25).
The IFITM5 mutation causing type V OI was quickly
speculated to have a gain-of-function mechanism because
of its N-terminal location and the hyperactive mineraliza-
tion traits of the type V OI phenotype (22, 23). Additional
indirect support for this interpretation was provided by
functional studies of normal BRIL, in which adenovirus-
mediated BRIL overexpression in UMR106 cells stimu-
lated mineralization and lentivirus-mediated Ifitm5
shRNA knockdown in MC3T3 cells inhibited mineraliza-
tion (19). Furthermore, comparison of the two IFITM5
mutations in primary patient osteoblasts in studies focus-
ing not on mineralization, but on pigment epithelium de-
Figure 3. Type I Collagen secretion and deposition A) Left, Representative western blot of type I procollagen secretion from control (C) or
patient 3 (OI V) OB stimulated with osteogenic media for up to 15 days, or Right, for 10 days with BMP2 treatment. patient 3 has decreased
collagen secretion and was equal with BMP2 treatment. B) Extracellular collagen matrix deposited by control (C) or patient 3 (OI V) OB. Normalized
densitometry of control and OI V
!
1(I) chains after balanced loading revealed a 3.5-fold decrease in mature cross-linked collagen, as well as a
decrease in crosslinked
"
-forms in acetic acid (AA) and pepsin (P) fractions. C) Steady-state type I collagen protein in osteoblasts from control (C)
and patient 3 (OI V). Migration of the
!
1(I) and
!
2(I) chains is normal in the patient. D) Immunofluorescence microscopy of collagen matrix
deposited in culture by control (C) and patient 3 (OI V) fibroblasts and osteoblasts. Type V OI matrix has patchy, parallel fibers in comparison to the
networked, well-formed fibers in control.
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rived factor (PEDF), also implicated a gain-of-function
mechanism for the IFITM5 mutation causing type V OI
(25). We demonstrated that the type V OI mutation had
complimentarity with the BRIL p.S40L substitution func-
tion, in that the BRIL p.S40L substitution decreased ex-
pression of PEDF by an unknown mechanism, while pri-
mary osteoblasts from type V OI patients have increased
PEDF expression and secretion (25), again supporting a
putative gain-of-function mechanism in type V OI. The
increased mineralization of type V OI has not been dem-
onstrated directly at the cellular level of primary patient
osteoblasts, nor has a relationship of the type V OI phe-
notype to type I collagen been demonstrated.
We report here 8 additional patients with type V OI and
the first studies of type V OI primary osteoblasts focused
on mineralization and differentiation. We confirmed ex-
pression of the mutant allele in patient osteoblasts and
fibroblasts, and the stability of the mutant transcript. Pa-
tients in all 5 families developed some of the clinical fea-
tures of type V OI, but there was considerable variation in
the features manifested and in their timing of appearance.
Even the elderly adult with severe OI, who died prior to
delineation of her OI type and mutation, had a history of
radial head dislocation. This clinical variability among
individuals heterozygous for the IFITM5 c.-14C!T mu-
tation has also been noted by others (24, 32).
IFITM5 transcripts were below the level of detectibility
by real-time RT-PCR in primary human osteoblasts, while
BRIL protein was detectable by Western blot at a compa-
rable level in type V and in control. Treatment with BMP2
to stimulate the osteogenic differentiation increased IF-
ITM5 transcripts in Type V OI and control osteoblasts to
a comparable level, and did not alter the relative levels of
transcripts for differentiation markers, providing reassur-
ance that BMP2 treatment had not fundamentally altered
type V OI osteoblast differentiation. In primary type V OI
osteoblasts, multiple markers of bone differentiation were
increased, including early (alkaline phosphatase), mid
(bone sialoprotein) and late (osteocalcin and osteopontin)
markers, consistent with a generalized acceleration in type
V bone cell differentiation.
Furthermore, the type V primary osteoblast studies
support a mechanism for type V OI which is specifically
collagen-related. The type V OI IFITM5 mutation is
shown here to cause decreased type I collagen expression
and secretion. Given the general increase in other osteo-
blast maturation markers, the decrease in type I collagen
expression stands out as reflecting a specific pathway
which is altered in the opposite direction to the generalized
increase in cell maturation. Consequent to the decreased
collagen secretion, Type V OI osteoblasts have decreased
collagen deposition into matrix and an altered deposition
pattern when fibrils are compared to control by fluores-
cent microscopy. This data supports the collagen-related
character of BRIL defects. The full pathway for BRIL func-
tioning in bone cells is an active area of investigation.
Finally, we demonstrated increased mineral deposition
by primary type V osteoblasts in culture. These data in-
dicate that increased osteoblast mineralization activity is
the underlying cause of the hyperactive mineralization
seen in patient traits such as hyperplastic callus and dense
metaphyseal bands. The studies with type V OI primary
osteoblasts presented here provide direct confirmation for
a cell-based gain-of-function mechanism, and support the
collagen-related character of BRIL defects.
In summary, these data demonstrate a gain-of-function
mechanism for the BRIL mutation causing type V OI at the
cellular level. In combination with the dominant inheri-
tance of type V OI, this data indicates that a BRIL protein
replacement strategy for treating type V OI is unlikely to
be successful. Instead, targeted therapy will need to focus
on the intracellular signaling pathways and partners of
BRIL to interfere with the mechanisms activating cell dif-
ferentiation and mineralization. Delineation of these path-
ways and their connection to PEDF (25) is under
investigation.
Acknowledgments
We thank the patients and their families for their dedicated long
term support of OI research. We thank the NICHD Microscopy
and Imaging Core in which the confocal microscopy was con-
ducted. This work was supported by NICHD intramural funding
(JCM). Authors’ roles: Study design: AR, ASB, AMB, and JCM.
Data collection: AR, ASB, AMB, WAC, AH, DC. Data analysis
and interpretation: AR, ASB, AMB, WAC, AH, DC, JC, SCH
and JCM. Drafting manuscript: JCM, AR, ASB. Revising man-
uscript content: AR, ASB, AMB, WAC, AH, DC, and JCM. Ap-
proving final version of manuscript: AR, ASB, AMB, WAC, AH,
DC, JC, SCH and JCM. JCM takes responsibility for the integrity
of the data analysis.
Address all correspondence and requests for reprints to: Com-
municating author: Joan C. Marini, MD, PhD, Chief, Bone and
Extracellular Matrix Branch, Building 10; Rm 10D39, 9000
Rockville Pike, Bethesda, MD 20 892, (T) 301–594-3418, (F)
301–480-3188, (e) oidoc@helix.nih.gov.
Disclosures Statement: The authors have nothing to disclose.
This work was supported by .
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