Article

The pro-Forms of Insulin-Like Growth Factor I (IGF-I) Are Predominant in Skeletal Muscle and Alter IGF-I Receptor Activation

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Endocrinology (Impact Factor: 4.5). 03/2013; 154(3):1215-24. DOI: 10.1210/en.2012-1992
Source: PubMed
ABSTRACT
IGF-I is a key regulator of muscle development and growth. The pre-pro-peptide produced by the Igf1gene undergoes several posttranslational processing steps to result in a secreted mature protein, which is thought to be the obligate ligand for the IGF-I receptor (IGF-IR). The goals of this study were to determine what forms of IGF-I exist in skeletal muscle, and whether the mature IGF-I protein was the only form able to activate the IGF-IR. We measured the proportion of IGF-I species in murine skeletal muscle and found that the predominant forms were nonglycosylated pro-IGF-I and glycosylated pro-IGF-I, which retained the C-terminal E peptide extension, instead of mature IGF-I. These forms were validated using samples subjected to viral expression of IGF-I combined with furin and glycosidase digestion. To determine whether the larger molecular weight IGF-I forms were also ligands for the IGF-IR, we generated each specific form through transient transfection of 3T3 cells and used the enriched media to perform kinase receptor activation assays. Compared with mature IGF-I, nonglycosylated pro-IGF-I had similar ability to activate the IGF-IR, whereas glycosylation of pro-IGF-I significantly reduced receptor activation. Thus, it is important to understand not only the quantity, but also the proportion of IGF-I forms produced, to evaluate the true biological activity of this growth factor.
The pro-Forms of Insulin-Like Growth Factor I (IGF-I)
Are Predominant in Skeletal Muscle and Alter IGF-I
Receptor Activation
Julia Durzyn´ ska,* Anastassios Philippou,* Becky K. Brisson,
Michelle Nguyen-McCarty, and Elisabeth R. Barton
Department of Anatomy and Cell Biology, School of Dental Medicine, and Pennsylvania Muscle Institute,
University of Pennsylvania, Philadelphia, Pennsylvania 19104
IGF-I is a key regulator of muscle development and growth. The pre-pro-peptide produced by the
Igf1gene undergoes several posttranslational processing steps to result in a secreted mature pro-
tein, which is thought to be the obligate ligand for the IGF-I receptor (IGF-IR). The goals of this study
were to determine what forms of IGF-I exist in skeletal muscle, and whether the mature IGF-I
protein was the only form able to activate the IGF-IR. We measured the proportion of IGF-I species
in murine skeletal muscle and found that the predominant forms were nonglycosylated pro-IGF-I
and glycosylated pro-IGF-I, which retained the C-terminal E peptide extension, instead of mature
IGF-I. These forms were validated using samples subjected to viral expression of IGF-I combined
with furin and glycosidase digestion. To determine whether the larger molecular weight IGF-I
forms were also ligands for the IGF-IR, we generated each specific form through transient trans-
fection of 3T3 cells and used the enriched media to perform kinase receptor activation assays.
Compared with mature IGF-I, nonglycosylated pro-IGF-I had similar ability to activate the IGF-IR,
whereas glycosylation of pro-IGF-I significantly reduced receptor activation. Thus, it is important
to understand not only the quantity, but also the proportion of IGF-I forms produced, to evaluate
the true biological activity of this growth factor. (Endocrinology 154: 1215–1224, 2013)
I
GF-I is critical for the growth and development of many
tissues. For skeletal muscle, IGF-I coordinates with addi-
tional growth factors to promote myoblast proliferation, dif-
ferentiation, and fiber formation during normal growth as
well as during regeneration after injury. Thus, IGF-I is a cen-
tral therapeutic targetfor enhancing muscle functionin aging
and disease. Several strategies have been employed to boost
IGF-I levels in muscle, including tissue-specific transgenic ex-
pression (1–3), viral-mediated gene transfer (46), and di-
rected recombinant IGF-I delivery (7, 8). Increasing IGF-I
levels can result in functional hypertrophy in young adult
animals, maintenance of mass and regenerative capacity in
senescent animals, and enhancement of muscle recovery to
counter acute and chronic damage.
IGF-I is produced from the Igf1 gene, which is more
than 90% conserved across species, and can undergo al-
ternative splicing at both the 5- and 3-ends to generate
multiple isoforms (Figure 1A) (reviewed in Ref. 9). Re-
gardless of the isoform transcribed, a pre-pro-peptide is
translated, which consists of a signal peptide directing se-
cretion, the mature IGF-I peptide, and a C-terminal ex-
tension called the E-peptide. After cleavage of the signal
peptide, the pro-IGF-I (mature IGF-I plus an E-peptide)
can be subjected to additional processing before secretion.
This includes cleavage of the E-peptide by intracellular
proteases to release mature IGF-I for secretion (10), main-
tenance of pro-IGF-I to be secreted without cleavage (11–
13), or N-glycosylation in the E-peptide of the predomi-
nant IGF-I isoform (IGF-IA) (14), and then secretion
(termed Gly-Pro-IGF-I). Hence, three forms of IGF-I pro-
tein could exist in the extracellular milieu: mature IGF-I,
nonglycosylated pro-IGF-I, and glycosylated-pro-IGF-I.
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2013 by The Endocrine Society
doi: 10.1210/en.2012-1992 Received September 27, 2012. Accepted January 17, 2013.
First Published Online February 13, 2013
* J.D. and A.P. contributed equally to this study.
Abbreviations: AAV, adeno-associated virus; ECM, extracellular matrix; GFP, green fluo-
rescent protein; IGFBP, IGF-binding protein; IGF-IR, IGF-I receptor; KIRA, kinase receptor
activation; PEG, polyethylene glycol; PNGase F, N-glycosidase F; TA, tibialis anterior.
GROWTH FACTORS-CYTOKINES
Endocrinology, March 2013, 154(3):1215–1224 endo.endojournals.org 1215
Page 1
(For this study, we will refer to nonglycosylated pro-
IGF-IA as pro-IGF-I.)
In a previous study, we compared the response of mus-
cle not only to the IGF-I isoforms (IGF-IA and IGF-IB), but
also to expression of mature IGF-I, generated by the in-
sertion of a premature stop codon before the E-peptide
sequence (15). Surprisingly, the overexpression of mature
IGF-I lacking any E-peptide did not promote muscle hy-
pertrophy in young mice, suggesting that the pro-IGF-I
forms are required for this effect. IGF-I acts predomi-
nantly via the IGF-I receptor (IGF-IR). Through the in-
herent tyrosine kinase activity of these receptors, ligand
binding mediates the signaling pathways necessary for cell
survival and growth. The general consensus is that the
mature IGF-I is the obligate ligand for these receptors, and
that the pro-IGF forms are precursors that must have the
C terminus removed before efficient receptor binding can
occur. The important residues for receptor binding are
distributed throughout the mature molecule (16, 17), and
none have been identified in the E-peptide, suggesting that
it is dispensable for receptor binding. However, the recent
development of polyethylene glycol (PEG)ylated mature
IGF-I protein (18) demonstrates that high molecular
weight modifications near the C terminus of mature IGF-I
Figure 1. IGF-I Forms in Muscle. A, Schematic presentation of alternative splicing of the Igf1 gene and the forms of IGF-I produced by the viral
constructs, with the recognition sites for the antibodies indicated. B, Immunoblotting shows the endogenous IGF-I forms in muscle (Mus). IGF-I
forms are evident upon long exposure for anti-IGF-I, with a mature IGF-I band at approximately 7 kDa, and multiple bands between 13 and 25
kDa. Blotting with anti-EA-peptide (EA) show that two bands (13 kDa, pro-IGF-I, and 20 kDa, gly-pro-IGF-I) are detectable with antibodies for EA
and IGF-I. A total of 100
g muscle protein per lane, 0.5 ng recombinant murine IGF-I (rIGF-I), and 20 ng recombinant EA-peptide were used as
positive controls. Lefthand lane (Coom) shows Coomassie-stained lane of gel containing muscle lysate. C, ELISA measurements demonstrate a 3-
to 4-fold increase in IGF-I content after viral delivery to murine skeletal muscle compared with uninjected muscles (Control). Results are shown as
mean SEM for N 4 muscles per condition. *P .05 injected vs control samples, P .05, comparisons of injected samples. D,
Immunoblotting for mature IGF-I and EA-peptide after viral expression of IGF-IA (IA) and IGF-ISt (ISt) shows that IGF-IA production results in two
pro-IGF-I forms, with no detectable mature IGF-I. Only mature IGF-I is produced after viral delivery of IGF-ISt, and IGF-I levels are below the level of
detection in control uninjected muscle lysates. Total protein was 20
g per lane. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MW,
molecular weight.
1216 Durzyn´ ska et al IGF-I Activity Is Dependent upon Its Form Endocrinology, March 2013, 154(3):1215–1224
Page 2
do not inhibit IGF-IR activation and also increase the half-
life of the growth factor. By extension, E-peptides found in
pro-IGF-I may not inhibit ligand-receptor interactions
and, in part, may act in a similar fashion to the PEG group.
Consistent with this model, we demonstrated that pro-
IGF-I produced by myoblasts has more efficient cell entry
into neighboring myoblasts compared with mature IGF-I
(19). More recently we have shown that the E-peptides
enhance IGF-I-mediated receptor signaling in vitro (20),
suggesting that even when the E-peptide is free, it may
modulate IGF-I activity. Therefore, the goals of this study
were to determine 1) the proportion of mature, pro-, and
glycosylated pro-IGF-I found endogenously, 2), the IGF-I
forms produced after expression of Igf1 in vitro and in
vivo, and 3), the apparent IGF-IR activity induced by these
forms.
Materials and Methods
Viral and plasmid constructs
Recombinant adeno-associated virus serotype 2/8 (AAV2/8),
produced at the University of Pennsylvania Vector Core and
harboring the Igf1 cDNA of murine class I IGF-IA and mature
IGF-I sequences, was used to express these IGF-I forms inskeletal
muscle, as described previously (15). Briefly, IGF-IA included the
sequence to encode the class I signal peptide, IGF-I, and the
EA-peptide, respectively. IGF-ISt lacked E-peptide sequence, and
a stop codon was inserted at the end of the mature IGF-I (A70X).
Plasmid DNA constructs containing the same Igf1 cDNAs
used for the viral constructs were also used for transfections of
3T3 cells, in order to produce media containing high levels of
IGF-I. Additional mutants were generated by site-directed mu-
tagenesis (QuikChange II; Stratagene, La Jolla, California) to
inhibit cleavage between mature IGF-I and the E-peptide and/or
N-glycosylation of the EA-peptide. Only the predominant iso-
form of IGF-I (IGF-IA) was mutagenized, because it represents
approximately 90% of the IGF-I produced by the muscle and
liver and harbors potential N-glycosylation sites (9). IGF-IA
cleavage inhibition required mutagenesis of three sites: K68G,
R71A, R77A (IGF-IKRR). Prevention of N-glycosylation in the
EA-peptide was achieved through the site-directed mutagenesis
of N92D (IGF-IN1), N100D (IGF-IN2), or both residues (IGF-
INN). Finally, mutant constructs blocking both cleavage and
N-glycosylation sites were generated to produce only pro-IGF-I
(IGF-IKRRNN). All cDNA constructs were inserted into the
NheI and XhoI restriction sites of pCMV.IRES.eGFP vector
(CLONTECH Laboratories, Inc, Mountain View, California)
for transient transfection as previously described (19).
Muscle AAV injections
All experiments were approved by the university animal care
committee. Viral injections of 1 10
11
particles diluted in 75–
100
L of PBS were performed into the anterior compartment of
one lower hind limb of anesthetized C57Bl/6 mice targeting the
tibialis anterior (TA) muscle. The contralateral limb received an
equal volume of PBS in the same manner as a control for the
injection procedure. After injection, mice were housed in the
animal facility until time of analysis. Mice (n 4 for each con-
struct) were 2–3 weeks of age at the time of injection. They were
killed 4 weeks after injection and exsanguinated, and the TA
muscle and other tissues were dissected and rapidly frozen in
liquid nitrogen for biochemical analysis. Serum was separated
from whole blood by centrifugation and stored at 80°C for
subsequent analysis.
Cell culture transfection
3T3 cells (1.2 10
6
) were grown on six-well plates (Falcon,
BD Bioscienses, Sparks, Maryland) in DMEM containing 10%
fetal bovine serum and supplemented with 100 U/mL ampicillin
and 100 U/mL streptomycin. Transient transfection was per-
formed using Lipofectamine 2000 (Invitrogen, Carlsbad, Cali-
fornia). For each transfection, cells were mixed with 0.5 mL of
Opti-MEM (Invitrogen) containing 6
g of plasmid DNA plus 8
L of Lipofectamine, and then 1.5 mL DMEM plus 10% fetal
bovine serum was added, and the cells were incubated for a total
of 4 hours. Cells were switched into minimal media (DMEM
supplemented with 100 U/mL ampicillin and 100 U/mL strep-
tomycin) for 24 hours after transfection. Controls included
transfection of empty vector (green fluorescent protein, GFP)
and no transfection (control). The next day, media from trans-
fected 3T3 cells was utilized for ELISA and immunoblotting
measurements of IGF-I, and for kinase receptor activation
(KIRA) assays (described below). 3T3 cell pellets were also re-
tained for determining transfection efficiency.
Immunoblotting analysis
Tissues were removed from liquid nitrogen storage and ho-
mogenized in 10 volumes/muscle wet weight of modified radio-
immune precipitation assay lysis buffer (50 mM TrisHCl [pH
7.4], 1% [wt/vol] Triton X-100, 0.25% sodium deoxycholate,
150 mM NaCl, protease, and phosphatase inhibitor cocktails
[P8340 and P5726, Sigma Chemical Co, St Louis, Missouri]).
Extracts from transfected cells were obtained by cell lysis in the
same RIPA buffer. Tissue homogenates and cell lysates were
centrifuged to pellet debris, and the total protein was measured
in the supernatant using the Bradford procedure (Bio-Rad pro-
tein assay; Bio-Rad Laboratories, Hercules, California). Media
from transfected cells were concentrated 10-to 20-fold using mi-
crocentrifugal filters (Microcon; Millipore, Billerica, Massachu-
setts) and with the addition of protease inhibitors (P8340,
Sigma), and then subjected to immunoblotting. In addition, me-
dia samples were subjected to serial dilution with DMEM and
then subjected to immunoblotting.
Equal amounts of protein from tissue and cell lysate, or equal
volumes of media, were subjected to SDS-PAGE using 16.5%
Tris/Tricine or 12.5% Tris/Glycine gels and transferred to poly-
vinylidene fluoride membranes (Immobilon-P; Millipore Corp).
Serial dilutions of recombinant murine IGF-I (R&D Systems,
Minneapolis, Minnesota) were loaded onto each blot (0.1–10 ng
per lane) in order to establish a standard curve for estimation of
the IGF-I content in the samples. The recombinant IGF-I immu-
noblot and ELISA measurements were consistent with the man-
ufacturers’ specifications, with slopes of the comparisons equal
to 1 and Y-intercept values less than 0.02. These standard curves
were utilized to estimate the IGF-I content in each sample lane.
The following primary antibodies were used for the immunode-
Endocrinology, March 2013, 154(3):1215–1224 endo.endojournals.org 1217
Page 3
tection of the IGF-I forms (Figure 1A): a rabbit polyclonal anti-
IGF-1Ea (20) (1:30 000 dilution), and a goat polyclonal anti-
mature IGF-I antibody (1:500 dilution) (AF791; R&D Systems).
Tissue lysates were also probed for glyceraldehyde-3-phosphate
dehydrogenase (1:5000) (antimouse glyceraldehyde-3-phos-
phate dehydrogenase, sc-32233, Santa Cruz Biotechnology,
Santa Cruz, California) to ensure equal protein loading. Con-
centrated media were also probed for IGF-binding protein
(IGFBP)-3 (1:1000) (R&D Systems, MAB775), to determine
whether the transfected constructs modulated the level of this
protein. Immunoblotting of cell pellets for transfection efficiency
utilized antibodies for GFP (1:1000) (catalog no. 2955, Cell Sig-
naling Technology, Beverly, Massachusetts), and
-tubulin (1:
4000) (T5168, Sigma). Immunoblotting of cell lysates for IGF-IR
phosphorylation used antibodies for P-IGF-IR (1:1000) (catalog
no. 407707, Calbiochem, San Diego, California) and IGF-IR
(1:1000) (sc-713, Santa Cruz Biotechnology). After washes and
exposure to secondary antibodies, specific bands were visualized
by x-ray film and by Image Quant LAS 4000 (GE Healthcare
Biosciences, Pittsburg, Pennsylvania), after incubation with an
enhanced chemiluminescent (ECL) substrate (Western lightning-
ECL, PerkinElmer, Waltham, Massachusetts). Analysis of band
intensity was performed by use of the associated Image Quant
software. Membranes were stained with Coomassie brilliant
blue R-250 after immunoblotting, and gels were stained with
colloidal Coomassie to confirm protein loading.
ELISA assays
Total IGF-I content in muscle protein extracts and in condi-
tioned media was determined by a standard sandwich ELISA
protocol using commercially available kit (MG100, R&D Sys-
tems) according to manufacturer’s recommendations and as pre-
viously described (4, 19). This kit detects total rodent IGF-I and
can also detect endogenous IGF-I production by C2C12 cells,
and there is no cross-reactivity or interference with IGF-II or
IGFBPs. The assay can detect IGF-I at 30–2000 pg/mL, with an
intraassay precision of 4.3% and an interassay precision of
5.9%. Data were acquired in duplicate using a SpectraMax M5
plate reader (Molecular Devices, Sunnyvale, California) at 450
nm, and the results were averaged.
Furin cleavage
To identify the potential processing products of each IGF-I
isoform, total protein lysates from AAV-injected TA muscles
from C57BL/6 mice were incubated with recombinant protease
furin (Recombinant Human Furin, 1503-SE-010, R&D Sys-
tems) according to manufacturer’s recommendations. Furin pro-
cesses proproteins by cleaving at specific motifs (ie, K-X-X-K-
X-X-R-X-X-R-X-X-R) usually residing at the end of their pro
regions. Equal amounts (30
g) of total protein from each AAV-
injected IGF-IA and IGF-ISt muscle lysate were incubated with
furin (20 U of furin per 30
g of each muscle lysate) overnight at
37°C. The corresponding volume of furin assay buffer (25 mM
Tris, 1 mM CaCl
2
, 0.5% [wt/vol] Brij-35]pH 9.0]) for each mus
-
cle lysate was used as control. After overnight incubation with
furin or furin assay buffer, muscle total protein extracts were
subjected to immunoblotting and to ELISA for IGF-I detection.
Deglycosylation
To identify the putative glycosylated (and nonglycosylated)
forms of IGF-I, total protein isolated from AAVIA-injected TA
muscles was incubated with N-Glycosidase F (PNGase F), which
cleaves between the innermost GlcNAc and asparagine residues
of high mannose and complex oligosaccharides from N-linked
glycoproteins. Aliquots containing 80
g of AAV IGF-IA muscle
protein extract were incubated with 2500 U of PNGase F (cat-
alog no. P0705S; New England Biolabs, Ipswich, Massachusetts)
for 3 hours at 37°C, according to manufacturer’s recommenda-
tions. The corresponding volume of PNGase assay reaction buf-
fer (G7 reaction buffer [10X], glycoprotein denaturing buffer
[10X], Nonidet P-40 [10%]) lacking the enzyme PNGase F was
used as control. After a 3-hour incubation with PNGase F or
reaction buffer, muscle protein samples were purified using de-
tergent removal spin columns (catalog no. 87777, Pierce Chem-
ical Co, Rockford, Illinois), and then incubated with furin, or
furin assay buffer (control) overnight at 37°C as described in the
previous section. The final reactions were then subjected to im-
munoblotting for IGF-I detection. The deglycosylation buffer
conditions were not compatible with ELISA measurements, and
therefore they were not pursued.
IGF-IR activation assay
To compare the potency of IGF-IR activation by the IGF-I
forms secreted from 3T3 cells after transfection, a KIRA assay
was performed as previously described (21) with a few altera-
tions. Briefly, 2.5 10
4
P6 cells, which overexpress IGF-IR (kind
gift from Dr Renato Baserga, Thomas Jefferson University, Phil-
adelphia, Pennsylvania) were seeded into 96-well plates. They
were maintained in growth media supplemented with 200
g/mL
G418. The cells were serum starved for 6 hours, and then treated
for 15 min with fresh media harvested from the transfected 3T3
cells. Controls included P6 cells treated with media alone, or with
recombinant IGF-I (0.5– 600 nM). For each KIRA experiment
replicates, one well of 3T3 cells was transfected with a single
IGF-I construct, giving rise to conditioned media containing the
IGF-I form produced by the construct. Each media sample was
tested in duplicate or triplicate on P6 cells. The P6 cells were lysed
and IGF-IR was captured onto an ELISA plate coated with an
antibody to IGF-IR (MAB1120, Millipore Corp.). A horseradish
peroxidase-conjugated antibody to phosphorylated tyrosines
(16 454, Millipore Corp.) and TMB substrate (N301, Thermo
Scientific, Rockford, Illinois) were used for colorimetric quan-
tification. Absorbance was read at 450 nm via the SpectraMax
M5 plate reader (Molecular Devices, Sunnyvale, California), and
values were averaged within each replicate. As validated previ-
ously (20) and in our hands, the assay had an intra-assay
precision of 4% for standards and 9% for test samples, and an
interassay precision of 12% for both standards and samples.
Calculation of relative activity for each replicate was per-
formed as described in Supplemental Appendix 1 published on
The Endocrine Society’s Journals Online web site at
http://endo.endojournals.org.
Statistical analysis
All statistical comparisons were performed in Prism (version
5.0c for Mac OS X, GraphPad Software, Inc, San Diego, Cali-
fornia). One-way ANOVA was employed to evaluate changes in
IGF-I content in both muscle lysates and cell-conditioned media
1218 Durzyn´ ska et al IGF-I Activity Is Dependent upon Its Form Endocrinology, March 2013, 154(3):1215–1224
Page 4
in all viral infection and transfection conditions, as well as the
KIRA assays, and was used to compare the apparent activity of
IGF-I. Where significant F ratios were found for main effects (P
.05), means were compared using Tukey’s post hoc tests. Un-
paired t-tests were used for comparisons of results from furin
experiments. KIRA dose-response curves were compared by an
extra sum-of-squares F test. All data are presented as mean
SEM. The level of statistical significance was set at P .05.
Results
The first goal of this study was to examine the forms of
IGF-I produced in skeletal muscle. We measured endog-
enous forms of IGF-I in muscle by immunoblotting high
amounts of protein (100
g) extracted from tissues. Mus-
cle had high molecular weight bands in addition to mature
IGF-I (Figure 1B). Two bands of approximately 13 and 20
kDa were also detected by the antibody for EA, supporting
that these bands were glycosylated pro and pro-IGF-I. We
do not know the identity of the higher molecular weight
bands (those greater than 25 kDa). Therefore, a significant
proportion of IGF-I found in naïve muscle is pro-IGF-I.
To improve IGF-I detection, we utilized muscle samples
subjected to viral mediated delivery of IGF-IA and mature
IGF-I (IGF-ISt). The levels of IGF-I in the AAV-injected
and control muscles were quantified by ELISA. Although
all injections resulted in significantly higher IGF-I content
than in uninjected controls, the production after viral de-
livery of IGF-ISt exceeded the levels produced by IGF-IA
by more than 40% (Figure 1C). To determine the forms of
IGF-I produced after viral delivery to murine skeletal mus-
cle, immunoblotting of muscle lysates (20
g total protein)
was performed with antibodies specific to IGF-I as well as
to the EA-peptide (Figure 1D). In contrast to the ELISA
measurements, the bands detected after AAV injection of
IGF-IA were of higher intensity than the bands found in
samples injected by AAV-IGF-ISt. Further, the predomi-
nant bands from IGF-IA samples migrated at a higher mo-
lecular weight (Figure 1D). IGF-ISt produced one band
approximately 7 kDa in size, which was the predicted size
for mature IGF-I, yet IGF-IA expression generated 13- and
17-kDa bands. Immunoblotting with an antibody recog-
nizing the EA-peptide was used (21) and also detected the
same bands (Figure 1C). Thus, most IGF-I species retained
in muscle after viral delivery of the full-length Igf1a open
reading frame are glycosylated pro- and pro-IGF-I forms,
not mature.
To clarify the identity of IGF-I forms produced by viral
expression of IGF-IA, muscle lysates were incubated with
furin and/or glycosidase to cleave mature IGF-I from the
E-peptide, and to separate the glycosylated side chains
from asparagine residues, respectively. This experiment
required levels of IGF-I produced by AAV, because en-
dogenous IGF-I levels were insufficient for immunoblot
detection after enzyme digestion. As shown in Figure 2A,
furin treatment increased the presence of mature IGF-I in
IGF-IA muscle lysates, and glycosidase collapsed the gly-
pro-IGF-IA forms to a single pro-IGF-I band. Furin cleav-
age efficiency was not affected by glycosylation, because
mature IGF-I was evident after furin treatment regardless
of whether the samples had been treated previously with
glycosidase. Importantly, it appeared that ELISA mea-
surements were most sensitive to mature IGF-I, not the
proforms (Figure 1), suggesting that the presence of the
E-peptide impaired the ability for the IGF-I antibody in the
ELISA to recognize the protein. To determine whether the
retention of the E-peptide impaired ELISA detection, mus-
cle lysates were incubated with furin, and then subjected
to IGF-I ELISA quantification. Comparison of the same
Figure 2. Identification of the Multiple Forms Produced by Viral
Delivery of the IGF-I Isoforms. A, Furin digest confirms that the higher
molecular weight forms produced by IGF-IA can be cleaved to produce
mature IGF-I. Deglycosylation of IGF-IA by PNGase treatment confirms
that the highest molecular weight bands produced by this isoform are
glycosylated. PNGase followed by furin digest results in increased
mature IGF-I in the sample. Blot shows the same muscle lysate sample
of 30-
g aliquots subjected to buffer only, furin alone, PNGase alone,
or PNGase followed by furin. B, ELISA quantification before and after
furin digestion overnight at 37°C shows the cleavage of the C-terminal
extension increases the detectable pool of IGF-I after viral delivery of
IGF-IA, but does not change the observed IGF-I in control muscle (no
viral delivery) nor after delivery of AAVIGF-ISt. *P .05 for
comparisons between apparent IGF-I levels before and after furin
digest. MW, molecular weight.
Endocrinology, March 2013, 154(3):1215–1224 endo.endojournals.org 1219
Page 5
sample before and after furin treatment showed that IGF-I
levels measured by ELISA increased after furin treatment
in IGF-IA muscles, even though immunoblotting did not
show an increase in total IGF-I (Figure 2B). In contrast,
IGF-ISt samples did not exhibit the discordance between
the ELISA measurements before and after furin treatment.
Thus, it appeared that the E-peptide in pro-IGF-I impaired
the ability to accurately measure IGF-I content under non-
denaturing conditions.
The multiple species of IGF-I produced in muscle raises
the question of which form(s) can activate the IGF-IR. We
moved to a cell-based system in order to control both the
form produced and to enable quantification of receptor
activation. Initially, we determined which asparagine res-
idue was utilized for glycosylation by replacing one or
both residues with aspartate. Immunoblotting of media
from 3T3 cells transfected with IGF-IA revealed a cluster
of glycosylated bands ranging from 17–20 kDa (Figure
3A). Transfection of IGF-IN1 or IGF-IN2 resulted in a
single 17-kDa band, whereas transfection of IGF-INN,
harboring a double mutation of N92 and N100, resulted
in a 13-kDa band lacking any glycosylation. Thus, both
asparagine residues could be utilized for glycosylation and
result in the 20-kDa band.
We expanded our sets of constructs to include those
with blockade of cleavage between mature IGF-I and the
E-peptide (IGF-IKRR) as well as blockade of cleavage and
glycosylation (IGF-IKRRN1, IGF-IKRRNN). As shown
in Figure 3A, IGF-ISt-transfected cells secreted only ma-
ture IGF-I. Cells transfected with IGF-IA secreted mature,
pro and gly-pro forms, and the presence of gly-pro-IGF-I
was eliminated by transfection of IGF-INN. Likewise, the
mutation of the cleavage sites between mature IGF-I and
the E-peptides by IGF-IKRR transfection did not produce
mature IGF-I. Glycosylation was preserved in the N1 mu-
tant of pro-IGF-I (IGF-IKRRN1 vs IGF-IKRRNN). Fi-
nally, mutation of both cleavage and glycosylation elim-
inated most mature IGF-I as well as gly-pro-IGF-I in the
media. These band patterns were consistent for N 4 sep-
arate series of transfections. The mean proportions of each
IGF-I form with respect to the specific construct aredisplayed
in Figure 3B. Immunoblotting of cell lysates for GFP con-
firmed that the variability in IGF-I secretion was not due to
the transfection efficiency (Supplemental Figure 1A).
Figure 3. Secretion of IGF-I Forms after Transient Transfection of IGF-I Constructs. A, Immunoblot detection of the IGF-I forms secreted (30
L
media per lane). IA, transfection of wild-type IGF-IA; ISt, IGF-ISt transfection; N1, transfection of IGF-IA with N92A mutation; N2, transfection of
IGF-IA with N100A mutation; NN, transfection of IGF-IA with N92A/N100A mutation; KR, blockade of cleavage site between mature IGF-I and the
E-peptide; KN1, blockage of cleavage and N92A mutation; KN, blockade of both cleavage and glycosylation; V, vector only; M, mock transfection.
B, The proportion of IGF-I forms secreted after transient transfection. C, ELISA measurements of IGF-I secreted after transient transfection. Data
are presented as IGF-I levels normalized to that in media from mature IGF-I transfections for four experiments. D, Quantification of total IGF-I
secreted after transient transfection based on immunoblot. *P .05 significantly different compared with mature IGF-I. E, Ratio of ELISA and
immunoblot quantification shows that presence of glycosylation causes an underestimation of IGF-I by ELISA. Data are presented as the sum of all
IGF-I bands produced by each construct normalized to bands from transfection of mature IGF-I in four experiments. *P .05 significantly different
compared with mature IGF-I. MW, molecular weight.
1220 Durzyn´ ska et al IGF-I Activity Is Dependent upon Its Form Endocrinology, March 2013, 154(3):1215–1224
Page 6
Comparison of ELISA and immunoblotting for estima-
tion of IGF-I in the enriched media reflected the observations
made in vivo. Specifically, ELISA measurements (Figure 3C)
underestimated the amount of IGF-I produced by constructs
when there was gly-pro-IGF-I present, compared with mea-
surements by immunoblotting (Figure 3, D and E). There-
fore, although ELISA is a robust measurement for mature
IGF-I, the presence of additional forms of IGF-I, specifically
glycosylated pro-IGF-I, impairs the accurate quantification
of the total IGF-I pool. This was particularly problematic for
IGF-IA and IGF-IKRR constructs.
To determine the potency for IGF-IR activation by each
IGF-I form, media from the transfected 3T3 cells were
utilized for KIRA assays (Figure 4A). The amount of IGF-I
per transfection was measured by immunoblotting taking
into account all IGF-I forms in each lane. Immunoblotting
was used instead of ELISA so that we did not underesti-
mate the amount of glycosylated pro-IGF-I in any sample.
Compared with mature IGF-I, we found that media con-
taining gly-pro-IGF-I had lower apparent IGF-IR activa-
tion. Specifically, IGF-IA and IGF-IKRR samples caused
30 8% and 27 7%, respectively, less IGF-IR phos-
phorylation per IGF-I molecule than mature IGF-I. In con-
trast, media containing predominantly pro-IGF-I after
IGF-INN and IGF-IKRRNN transfections had equivalent
IGF-IR phosphorylation to mature IGF-I. To determine
whether the difference in IGF-IR phosphorylation was af-
fected by a change in binding proteins, media content of
IGFBP3 was evaluated by immunoblotting, because 3T3
cells are known to secret IGFBP3 (22). However, there was
no detectable IGFBP3 in the concentrated media (Supple-
mental Figure 1B). Thus, it appeared that gly-pro-IGF-I
was less able to activate the IGF-IR independent of
changes in binding protein level.
Because many of the enriched me-
dia samples contained multiple
forms of IGF-I, we took advantage of
knowing the proportion of each spe-
cies (Figure 3B) and the apparent re-
ceptor activation to calculate the
contribution of each IGF-I form to
the final IGF-IR response by setting
up a series of simultaneous equations
and solving for apparent activity of
mature, pro-, and gly-pro-IGF-I in
each replicate (Supplemental Appen-
dix 1). As shown in Figure 4B, we
found that pro-IGF-I could, in the-
ory, activate the IGF-IR as well as
mature IGF-I, yet gly-pro-IGF-I was
2-fold less potent than mature IGF-I.
Thus, our calculated comparisons
supported that both pro-IGF-I and mature IGF-I were po-
tent ligands for the IGF-IR.
Even though transfections of many of our constructs
resulted in a mixed population of IGF-I forms (Figure 3B),
transfection of IGF-IStop and IGF-IKRRNN routinely
produced pure mature and pro-IGF-I, respectively. Be-
cause the initial measurements were not at maximal acti-
vation levels, we extended the analysis of mature IGF-I and
pro-IGF-I activity to cover a wide range of concentrations.
We generated a dose-response curve of IGF-IR activation
for mature and pro-IGF-I. First, we concentrated or di-
luted the conditioned media produced after transfection
into 3T3 cells and estimated the IGF-I content by immu-
noblotting and ELISA (Figure 5, A and B). Although both
comparisons were close to unity, they were significantly
different, with mature IGF-I values higher when quanti-
fied by immunoblot than by ELISA (both mature bands
used for quantification), and with pro-IGF-I values lower
by immunoblot than by ELISA. This comparison sug-
gested that it was the state of glycosylation that impaired
accurate ELISA measurements of IGF-I when all forms
were present.
We confirmed that these forms caused phosphorylation
of the IGF-IR by immunoblotting, and saw that both gen-
erated robust phosphorylation of IGF-IR (Figure 5C). Ali-
quots of the same media were used in a KIRA assay, and
we found that exposure of cells to pro-IGF-I resulted in
similar IGF-IR activity as mature IGF-I (Figure 5D). The
maximum activation we measured for mature IGF-I was
10 1, for pro-IGF-I was 14 3, and for recombinant
murine IGF-I was 17 2 (mean SEM for four exper-
iments), which were not significantly different from each
other. To exclude that additional factors in the condi-
tioned media may have altered activity, the same assays
Figure 4. Comparison of IGF-IR Activation by IGF-I Forms. A, KIRA assay IGF-IR phosphorylation
levels by IGF-I in enriched media quantified by immunoblotting. Results are compared with
mature IGF-I activation within each KIRA experiment, where each sample was measured in
triplicate. Data shown are mean SEM for four experiments. B, Calculated contribution of
mature IGF-I, pro-IGF-I, and gly-pro-IGF-I to IGF-IR activation. Pro-IGF-I displays equivalent
activation of the receptor compared with mature IGF-I, whereas gly-pro-IGF-I has a significantly
reduced ability to activate the IGF-IR. *P .05 for comparisons between conditions.
Endocrinology, March 2013, 154(3):1215–1224 endo.endojournals.org 1221
Page 7
were performed on conditioned media obtained from 3T3
cells transfected with vector only. We found that vector-
only samples did not increase IGF-IR activity even when it
was concentrated 4-fold (Supplemental Figure 2) in con-
trast to IGF-I containing samples, nor did the addition of
concentrated media to recombinant IGF-I impair activity
(data not shown). This supports that the forms of IGF-I
were the only agents in the conditioned media that acti-
vated the IGF-IR. Based on the results of this study, we
assert that both mature and pro-IGF-I are ligands for the
IGF-IR. In addition, because gly-pro-IGF-I is a less potent
ligand for the receptor, we propose that it may serve as a
reservoir for IGF-I that can be stored in the extracellular
matrix (ECM) until needed, when the glycosylated C ter-
minus is removed to release mature IGF-I.
Discussion
The goals of this study were to identify the forms of IGF-I
produced in muscle, and to clarify which of these forms
were able to activate the IGF-IR. We found that untreated
muscle retains predominantly the glycosylated pro- and
pro-IGF-I forms, and when Igf1a is overexpressed by viral
delivery, both pro- and glycosylated pro-IGF-I are present
at high levels, but there is little mature IGF-I in either case.
Cell-based assays for IGF-IR activation demonstrated that
pro-IGF-I was as efficient at receptor activation as mature
IGF-I, yet gly-pro-IGF-I was significantly less effective. To
our knowledge, this is the first study to distinguish the
potency of the different species of IGF-I produced, and
helps not only to explain our previous observations, but
also establishes new potential ways to optimize IGF-I
activity.
The additional production of gly-pro-IGF-I by IGF-IA
expression may act as a ligand reservoir, although this was
not directly addressed in this study. Because it is less effi-
cient at activating the IGF-IR, the glycosylation of the
E-peptide may inhibit receptor binding. Although the crys-
tal structure of mature IGF-I, as well as mutational anal-
ysis of the protein, clearly delineates the surface in mature
IGF-I that is important for ligand binding (21, 23), the
structure of the E-peptide extension has not been resolved.
Figure 5. Quantification and Activity of IGF-I Forms. A, Immunoblot of concentrated conditioned media from 3T3 cells transfected with IGF-IStop,
IGF-IKRRNN (Pro-IGF-I), or vector only (Vect). Media were concentrated using microcentrifugal filters and then serially diluted. Serial dilution of
murine recombinant IGF-I was used as a standard. B, Comparison of ELISA and immunoblotting measurements for the same IGF-I samples. C,
Immunoblot for IGF-IR phosphorylation after 20 minutes exposure to IGF-IStop media (ISt), pro-IGF-I media (Pro), or 2 nM murine recombinant IGF-
I. D, KIRA assay for media from IGF-I Stop transfected 3T3 cells (Mature IGF-I), media from IGF-IKRRNN transfected 3T3 cells (Pro IGF-I), and murine
recombinant IGF-I (Recomb IGF-I), based on the amount of IGF-I in each sample (by blot). Both bands were combined for mature IGF-I levels. Data
are generated from three independent transfections for each construct, and three independent KIRA assays, as well as immunoblot quantification
for all samples as in panels A and B. Each point represents the mean of duplicate measurements for any given sample. The fold change increase in
IGF-IR phosphorylation is calculated from raw OD
450
values for each sample compared with negative control (no IGF-I added).
1222 Durzyn´ ska et al IGF-I Activity Is Dependent upon Its Form Endocrinology, March 2013, 154(3):1215–1224
Page 8
Our KIRA measurements suggest that the EA-peptide gly-
cosylation covers the ligand-binding site for IGF-I to the
receptor. However, whereas the nonglycosylated E-pep-
tide in the pro-IGF-I species may also have a similar con-
formation, it does not seem to impede receptor phosphor-
ylation. The recent development of PEGylated IGF-I also
supports that a C-terminal extension does not necessarily
block ligand binding, which is consistent with our obser-
vations (18). However, the affinity of the PEG-IGF-I was
less than that of mature IGF-I, suggesting that it may be
more similar to gly-pro-IGF-I.
Although the in vivo measurements show stable accu-
mulation of the pro- and gly-pro-IGF forms in the muscle
lysates, we cannot confirm that the location of the IGF-I
pool. Classically, IGF-I posttranslational processing in-
cludes intracellular cleavage of mature IGF-I from the E-
peptide, leading to mature peptide secretion, and an un-
known destination for the E-peptide (10). However, the
highly orchestrated process of glycosylation poses another
order of complexity onto IGF-I processing. The forms we
have observed appear to be stable intermediates or final
products of posttranslational processing, given that furin
and glycosidase digests confirm the identity of each band.
Further, our cell-based measurements clearly show that all
IGF-I species can be secreted, and substantiate the previ-
ous observations of pro-IGF-I in cell media (10, 12, 13,
19). In fact, most of the IGF-I is pro-IGF-I or gly-pro-IGF-I
in the media from IGF-IA-transfected cells, suggesting that
only a small portion of IGF-I cleavage occurs intracellu-
larly, or that cleavage occurs outside of the cell. In addi-
tion, comparison of the media from IGF-IA and IGF-INN
transfections show that blockade of glycosylation did not
affect the final amount of mature IGF-I secreted (Figure
3B). This suggests that the cell may reserve a portion of the
nascent IGF-I propeptides for intracellular cleavage, and
the rest is directed for glycosylation and/or secretion. In
muscle, these pro-forms could be stored in the ECM for
subsequent activity by cleavage, which has been proposed
recently (24). However, the extent of glycosylation or
cleavage may vary across different cell types. These pos-
sibilities will require further experiments to delineate
where each form of IGF-I resides in the muscle.
The murine IGF-IA has two potential N-glycosylation
sites, N92 and N100, both of which follow the consensus
sequence NXS/T, where X can be any residue except pro-
line. In humans and nonhuman primates, only the se-
quence surrounding N92 is conserved (25), suggesting
that if the same glycosylation patterns occurred in all spe-
cies, N100 may not be used. Therefore, we anticipated that
mutation of N92 would block glycosylation, whereas mu-
tation of N100 would not. However, site-directed mu-
tagenesis of either asparagine only removed the presence
of the faint bands above 17 kDa, and instead the migration
patterns of N92A and N100A mutants were indistinguish-
able. Only when both sites were mutated did all gly-pro-
IGF-I bands disappear. Based on these results, it appeared
that both residues could be glycosylated in mouse 3T3
cells, and therefore in order to generate pro-IGF-I for the
KIRA assay, we used the double mutant. Although not
directly addressed in this study, differential glycosylation
of IGF-I may occur in different tissue types and provide
another point for regulating stability and/or local reten-
tion of the growth factor. Further studies are needed to
address the tissue specificity of the posttranslational mod-
ifications of IGF-I, and to determine whether these mod-
ifications change IGF-I actions in vivo.
How the pro- and gly-pro-IGF forms reside in the mus-
cle matrix is an open question. Like the receptor-binding
assays, these forms may be able to associate with IGFBPs,
which are known stabilizers of IGF-I, but this was not
addressed in the current study. Although there was no
apparent change in the level of IGFBP3 in conditioned
media, this does not address differential binding affinity of
the various forms of IGF-I to IGFBPs. One advantage of
utilizing conditioned media is that the native glycosylation
pattern and folding of IGF-I is preserved. However, the
disadvantage of thisapproach is that additional factors are
also present to alter receptor activation, and measurement
of IGF-I content must be done indirectly. As such, we can-
not distinguish between a model in which pro-IGF-I has
lower affinity for IGFBPs leading indirectly to strong re-
ceptor activation vs a model in which pro- and mature
IGF-I have similar potencies. Future studies to address
these distinctions will require pure preparations.
The glycosylated residues may also associate directly
with the ECM, serving as an alternate additional reservoir
of this growth factor. Cleavage of the entire E-peptide by
extracellular proteases could then release active mature
IGF-I for receptor binding when needed. There is evidence
for candidate furin-like proteases residing in the ECM that
could cleave mature IGF-I from the E-peptide. The pro-
protein convertase subtilisin/kexin type 6, commonly
known as PACE4, is a likely candidate to cleave pro-IGF-I,
given the importance of this proprotein convertase for
muscle differentiation (26), and the fact that itcan perform
the same reaction intracellularly (10). Alternatively, the
glycosylation could be clipped from the core protein, re-
leasing pro-IGF-I for receptor activation. However, there
is little evidence for extracellular glycosidases that could
achieve this alternative path of regulation and remove gly-
cosylation from the IGF-I C terminus.
In summary, we have identified multiple species of
IGF-I that are produced by muscle both endogenously and
after viral expression of IGF-IA. The major forms that
Endocrinology, March 2013, 154(3):1215–1224 endo.endojournals.org 1223
Page 9
accumulate in the tissue are not mature IGF-I, but instead
pro-IGF-I and gly-pro-IGF-I. In addition, we have shown
that the species of IGF-I produced by the Igf1a isoform
have differential ability to activate the IGF-IR, where pro-
IGF-I and mature IGF-I are more efficient at receptor ac-
tivation than glycosylated pro-IGF-I. In future studies of
IGF actions in muscle, it will be important to account for
the forms produced and how they behave in physiologic
and pathologic situations.
Acknowledgments
We thank R. Baserga for providing the P6 cells. We also thank R.
Ma for generating the mutant IGF-I constructs. We are indebted
to Drs. K. Speicher and H. Tang for extensive advice on quan-
tification of IGF-I.
Address all correspondence and requests for reprints to: Elisa-
beth R. Barton, PhD, 240 South 40th Street, 441A Levy Building,
Philadelphia, PA 19104. E-mail: erbarton@dental.upenn.edu.
This work was supported by National Institutes of Health
Grant AR057363 (to E.R.B.). B.K.B. was supported by a fellow-
ship (AR053461) from the Pennsylvania Muscle Institute, Uni-
versity of Pennsylvania, Philadelphia, Pennsylvania.
Current address for A.P.: Department of Experimental Phys-
iology, Medical School, National & Kapodistrian University of
Athens, Goudi-Athens, Greece.
J.D. is a visiting scholar whose permanent appointment is the
Department of Molecular Virology, Institute of Experimental
Biology, Adam Mickiewicz University, Poznan´ , Poland.
Disclosure Summary: The authors have nothing to disclose.
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