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Endocrinology, doi:10.1210/en.2007-0274
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Endocrinology Vol. 148, No. 12 6176-6185
Copyright © 2007 by The Endocrine Society

Transgenic Overexpression of Pregnancy-Associated Plasma Protein-A Increases the Somatic Growth and Skeletal Muscle Mass in Mice

Mark Rehage, Subburaman Mohan, Jon E. Wergedal, Blake Bonafede, Kiet Tran, Diana Hou, David Phang, Ashok Kumar and Xuezhong Qin

Musculoskeletal Disease Center (M.R., S.M., J.E.W., B.B., K.T., D.H., D.P., X.Q.), Laboratory for Skeletal Muscle Physiology and Neurobiology (A.K.), Jerry L. Pettis Memorial Veterans Affairs Medical Center, and Departments of Medicine (S.M., J.E.W., A.K., X.Q.), Biochemistry (S.M.), Physiology (S.M.), Loma Linda University, Loma Linda, California 92357

Address all correspondence and requests for reprints to: Xuezhong Qin, Ph.D., Musculoskeletal Disease Center, J. L. Pettis Veterans Affairs Medical Center (151), 11201 Benton Street, Loma Linda, California 92357. E-mail: xuezhong.qin{at}med.va.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although IGFs are indispensable to skeletal muscle development, little information is available regarding the mechanisms regulating the local action of IGFs in skeletal muscle tissues. Here we tested the hypothesis that pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, promotes skeletal muscle formation in vivo through degrading IGF binding proteins (IGFBPs), which increases the bioavailability of IGFs. Expression of PAPP-A is significantly increased in muscle five days after muscle injury in mice. Targeted overexpression of PAPP-A using a muscle-specific promoter significantly increased the prenatal/postnatal growth, skeletal muscle weight, and muscle fiber area in mice. These anabolic effects were reproduced using F2/F3 progeny. Free IGF-I concentration was severalfold higher in the conditioned medium (CM) of ex vivo cultured muscle from the transgenic mice, compared with the wild-type littermate muscle. Accordingly, the proliferation of C2C12 myoblasts was significantly increased in the presence of CM from cultured skeletal muscle of the transgenic mice, compared with the controls. This observed increase in myoblast proliferation was abolished on addition of noncleavable IGFBP-4 peptide, which reduced free IGF-I concentration back to the basal level of the wild-type CM. Furthermore, proliferation and differentiation of C2C12 myoblasts was increased by transient overexpression of proteolytically active PAPP-A but not by inactive mutant PAPP-A (E483/A). Collectively, we identified PAPP-A as a novel regulator of prenatal/postnatal growth and skeletal muscle formation in vivo. Moreover, our studies provide the first experimental evidence that IGFBP degradation is a key determinant in modulating the local action of IGFs in muscle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IMPAIRMENT IN SKELETAL myogenesis has a significant impact on human health in a variety of physiological and pathophysiological conditions. For example, sarcopenia (loss of skeletal muscle mass and strength in the elderly) not only limits physical mobility but also increases susceptibility to muscle injury and bone fracture, a result of increased risk of falls (1, 2, 3, 4, 5). Skeletal muscle function becomes significantly compromised in certain disease states such as diabetes (6) and sepsis (7) or from some medical treatments, such as glucocorticoid therapy (8). In addition to pathological conditions, massive loss of skeletal muscle tissue after a traumatic injury could lead to disability or even death (9). Identifying molecules that promote muscle formation and understanding their mechanism of action are critical for future development of therapeutics for skeletal muscle-related disorders.

IGFs play a central role in the regulation of skeletal muscle development, maintenance, and regeneration (10, 11). The actions of IGFs in vitro and in vivo are modulated by six high-affinity IGF binding proteins (IGFBPs) (12, 13, 14). Most of the studies concerning the role of IGFBPs in myogenesis have primarily been restricted to evaluating the effects of exogenously added IGFBPs on myoblast proliferation and differentiation (10, 15, 16, 17, 18). Thus far, the role of endogenously produced IGFBPs has not been evaluated in skeletal muscle cells. Because the bioavailability of IGFBPs is determined by not only their rate of synthesis but also rate of degradation, research in the past several years has been focused on the proteases that specifically cleave IGFBPs. One of the major breakthroughs in this research area has been the identification of the pregnancy-associated plasma protein-A (PAPP-A) as a protease for IGFBP-2, -4, and -5 (19, 20, 21). PAPP-A is a secreted glycoprotein discovered in the blood of pregnant women (22). In circulation, the majority of PAPP-A exists as the 450-kD complex, formed by a covalent interaction between PAPP-A and the proform of eosinophil major basic protein (proMBP) (23). This PAPP-A-proMBP complex consists of two 200-kDa PAPP-A subunits disulfide bound to each of two mutually disulfide-bridged 50- to 90-kDa proMBP subunits. However, PAPP-A secreted by cultured cells exists as a free form, migrating as a band of 400-kDa protein under nonreducing conditions (19, 24). In addition to the protease domain, PAPP-A also contains several other functional domains such as the five complement control protein modules (1, 2, 3, 4, 5) and two Lin12-Notch repeats (25). To date, the functional significance of these additional domains in the biological actions of PAPP-A is not clear.

Because PAPP-A and its proteolytic target IGFBPs are expressed by myoblasts in vitro and skeletal muscle in vivo, PAPP-A could act as an important regulator of skeletal muscle development. This contention is supported by our recent findings that the exogenous addition of recombinant PAPP-A, or overexpression of PAPP-A, enhanced the proliferation and differentiation of C2C12 myoblasts (26). In this study, we tested the hypothesis that muscle-specific transgenic overexpression of PAPP-A will increase skeletal myogenesis in mice. Consistent to our hypothesis, our data show that PAPP-A transgenic mice exhibit a dramatic increase in prenatal/postnatal growth and skeletal muscle mass. Furthermore, we show that the stimulatory effects of PAPP-A on myoblast proliferation and differentiation are intrinsic to its proteolytic activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM was purchased from Invitrogen (Carlsbad, CA). The 6x histidine-tagged recombinant IGFBP-4, amino acid 121–142-deleted IGFBP-4 peptides, and recombinant FLAG-PAPP-A were prepared as previously described (26, 27). Purified polyclonal rabbit anti-human (h) PAPP-A IgG and rabbit control IgG were purchased from Dako Corp. (Carpinteria, CA). M2 FLAG antibody, laminin antibody, horse serum, and fetal calf serum (FCS) were from Sigma Chemical Co. (St. Louis, MO). Creatine kinase assay kit was obtained from Stanbio Laboratory (Boerne, TX). Free IGF-I ELISA kit was purchased from Diagnostic Systems Laboratories (Webster, TX). Recombinant human IGFBP-2, IGFBP-5, IGF-I, and IGF-II were purchased from GroPep (Australia). All other chemicals were of reagent grade and were obtained from Sigma.

PAPP-A plasmids
Construction of hPAPP-A (1–1547)/pFLAG plasmid has been previously described (26). The mutant PAPP-A (E483/A) plasmid was prepared as described below. A 1918-bp PAPP-A cDNA containing the E483 codon was prepared by PCR using pfu DNA polymerase, the PAPP-A (1–1547)/pFLAG plasmid, and primers given in Table 1Go. The PCR product was cloned into pCR*Blunt II TOPO vector (Invitrogen) and designated PAPP-A 1.9 kb/ZeroBlunt. The codon E483 (GAG) was mutated to the codon for A483 (GCG) by PCR using the PAPP-A 1.9 kb/ZeroBlunt as template and the primers given in Table 1Go. The PCR product was treated with DpnI, purified, incubated with T4 ligase, and transformed into methylation-deficient dam-/dcm-Escherichia coli (New England Biolabs, Ipswich, MA). After confirming the presence of the E483/A mutation and the absence of any unwanted mutations in the 1.9-kb PAPP-A cDNA sequence, the 1765-bp PAPP-A cDNA containing the E483/A mutation was isolated by digesting this intermediate plasmid with NarI and ClaI and ligated into the nonmethylated PAPP-A (1–1547)/pFLAG plasmid, in which the native NarI-ClaI fragment had been removed. The resulting plasmid was designated PAPP-A (483E/A)/pFLAG and used for transfection.


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  		TABLE 1. Primers used for genotyping and mRNA quantitation

 
Transfection and assay for myoblast proliferation and differentiation
C2C12 myoblasts (American Type Culture Collection, Manassas, VA) were grown at 37 C in a CO2 incubator in DMEM containing 10% FCS supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. C2C12 myoblast transfection was done using the Effectene transfection reagent following the suggested protocol by the manufacturer (QIAGEN, Valencia, CA). Proliferation of C2C12 myoblasts was measured using AlamarBlue dye and confirmed by measuring the total protein content in cell lysates as described (26). Differentiation of C2C12 myoblasts was induced by replacing the medium with differentiation medium (2% heat-inactivated horse serum in DMEM) for 72–96 h. Creatine kinase (CK) activity in cell lysates was measured as described (26) to study muscle differentiation.

Muscle crush injury in mice
The protocol for skeletal muscle crush injury is based on published studies (28) and approved by the Institutional Animal Care and Use Committee of Loma Linda Veterans Affairs Medical Center. The mice were anesthetized by sc injection of ketamine/xylazine and received pain reliever (buprenorphine) before surgery and two times per day for 2 d thereafter. A 5-mm skin incision was made using a sterile sharp scalpel, and the tibialus anterior (TA) muscle was separated from other muscles using a 28-gauge needle. Pressure was applied to the entire TA muscle using forceps. The skin was closed by simple interrupted suture. The same skin incision was made on the right hind limb: TA muscle was exposed but not crushed, and the skin was sutured and then served as control. After 3, 5, and 14 d of muscle injury, mice were killed by carbon dioxide inhalation followed by decapitation. Injured (left) and uninjured TA (right) muscles were excised, cleaned of adipose and connective tissue, and used for RNA isolation and quantitation of PAPP-A mRNA.

Generation of PAPP-A transgenic mice
Development of PAPP-A transgenic mice and subsequent tissue collection/phenotypic analyses were carried out according to protocols approved by Institutional Animal Care and Use Committee of Loma Linda Veterans Affairs Medical Center. The 4.6-kb human FLAG-PAPP-A cDNA was ligated at the NotI site of pBSX-HSAvpA plasmid (provided by Dr. Jeff Chamberlain, School of Medicine, University of Washington, Seattle, WA), which contains a 2.3-kb human skeletal {alpha}-actin promoter. For the purpose of transgenic plasmid linerization, the KpnI site before the actin promoter sequence in the pBSX-HSAvpA plasmid was replaced with a PvuI site using DNA linkers. The transgenic plasmid was then digested with PvuI, and the 8-kb fragment containing the PAPP-A expression cassette was purified and injected into the pronuclei of fertilized zygotes from C57BL/6J x CBA/CA mice at the Core Transgenic Mouse Facility of the University of Southern California. F1 generation mice were produced by breeding the transgenic founders with C57BL/6J mice. F2 generation mice were produced by breeding a male F1 PAPP-A transgenic mice with female C57BL/6J mice. The genotype of the mice was determined by PCR analysis of tail DNA using primers given in Table 1Go. Expression of PAPP-A in the skeletal muscle of transgenic mice was confirmed by semiquantitative RT-PCR and Western blot analysis of conditioned medium (CM) from cultured muscle at the end of the experiments.

Dual x-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT) analysis
Lean body mass and fat mass were determined under general anesthesia by DEXA using the PIXImus instrument (Lunar Corp., Madison, WI) as described (29). A transgenic mouse and a littermate control mouse were analyzed simultaneously for each scan. The forearm cross-sectional area (CSA), an indicator of the muscle size, was analyzed by pQCT (Stratec XCT 960M; Norland Medical Systems, Ft. Athinson, WI) on live mice as described (30). The anesthetized mouse was placed on a holding platform with the right forearm straightened and the flexor surface of the manus securely taped down. The length of the ulna was determined by using the bone scan from the pQCT software. A voxel size of 0.07 mm and a slice thickness of 0.5 mm were set for analysis of the mid-forearm CSA.

Histomorphometric of skeletal muscle sections
The CSA of the TA muscle and CSA of myofiber were determined by histomorphometry as described (31). Briefly, tendon-to-tendon TA muscles were isolated, weighed, fixed in 10% formalin, and embedded in paraffin. Five-micromole sections were cut at the midbelly of each TA muscle and subjected to either hematoxylin and eosin (H & E) staining or immunohistochemical staining for laminin using laminin antibody (Sigma). The TA muscle CSA and myofiber area in the midsections were determined under x2 and x20 magnification, respectively. The average myofiber size was calculated from the 10 fields of view in each section using an Oylmpus BX60 microscope and the Osteomeasure software from OsteoMetrics Inc. (Decatur, GA).

Semiquantitative PCR and quantitative real-time PCR analysis
Total RNA was isolated from the TA muscle using a RNeasy kit (QIAGEN). Reverse transcription was conducted using a commercial kit using 300 ng total RNA and random hexamers. Reaction without reverse transcriptase served as a negative control. Semiquantitative PCR was carried out using 5 µl of 5x diluted RT product, Taq DNA polymerase (New England Biolabs), and primers designed specifically for genes of interest (Table 1Go) under the following conditions: an initial 94 C for 10 min, 94 C for 45 sec, 55 C for 30 sec, and lastly 72 C for 30–60 sec. Based on the predetermined PCR kinetics, the number of PCR cycles was set as 21 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 33 for PAPP-A and IGF-I, respectively.

Real-time PCR analysis was carried out using Brilliant Syber Green QPCR master mix (Stratagene, La Jolla, CA) under conditions described for semiquantitative PCR, except that the cycle number was increased to 40. Cot curve values were calculated using the OpticonMonitor software (Promega, Madison, WI). Mouse GAPDH expression was used to adjust the mRNA level for each gene of interest.

Ex vivo muscle culture and analyses
Production and activity of hPAPP-A protein in each transgenic line was confirmed by analyzing the CM of ex vivo cultured muscle derived from 6-month-old F1 generation mice. Skeletal muscle (~50 mg) was cultured individually in 2 ml DMEM/10% FCS for 43 h. CM, after adjustment for muscle protein, was subjected to immunoblot analysis using either FLAG antibody or hPAPP-A antibody as described previously (24, 26).

The activity of PAPP-A in the CM was determined by incubating 50 ng IGFBP-4 with 2–10 µl CM in the presence of 25 ng IGF-II. After 2–4 h incubation, proteolysis of IGFBP-4 was evaluated by 125I-IGF-II ligand blot analysis as described previously (20).

To determine whether the increased IGFBP-4 proteolysis is contributed to the transgene product, human PAPP-A, instead of other nonspecific proteases, IGFBP-4 proteolysis was determined in the presence of control rabbit IgG or rabbit PAPP-A neutralization IgG. IGFBP-4 (50 ng) and IGF-II (25 ng) were incubated at 37 C for 3 h with pooled transgenic CM (1.5 µl) in the presence of control IgG (20 µg) or anti-PAPP-A IgG (20 µg). IGFBP-4 proteolysis was then evaluated by IGF-II ligand blot analysis (20).

Free IGF-I concentration was determined using a commercial kit with a sensitivity of detecting 0.1 ng/ml IGF-I (Diagnostic Systems Laboratories). This assay was developed to measure human free IGF-I but can also be applied to measure bovine-free IGFs because human IGF-I and bovine IGF-I are identical (26, 32). Because muscles (or murine C2C12 myoblasts) were cultured in the presence of 10% FCS, the free IGF-I measured by this assay is of bovine origin. The assay was validated in our laboratory by quantitation of free IGF-I in samples in the presence or absence of excess IGFBP-4.

Statistical analysis
Results are expressed as mean ± SEM and statistically analyzed by Student’s t test or ANOVA. A value of P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PAPP-A expression in skeletal muscle is up-regulated after muscle injury
Our recent studies demonstrate that PAPP-A enhances myoblast proliferation and differentiation in vitro (26). These intriguing findings led us to examine the role of PAPP-A in the regulation of skeletal muscle formation in vivo. If locally produced PAPP-A plays an important role in skeletal myogenesis, one would expect PAPP-A expression in skeletal muscle to be modulated under situations that affect skeletal myogenesis. To address this issue, we compared the level of PAPP-A expression in the TA muscle after muscle injury. An increase in PAPP-A mRNA level was evident at d 3. By d 5, the PAPP-A mRNA level was significantly higher in injured muscle, compared with uninjured muscle (Fig. 1Go). By d 14, PAPP-A mRNA level decreased to a level below that measured in the undamaged muscle (Fig. 1Go). It is important to note that the currently available human PAPP-A antibodies do not recognize mouse PAPP-A. Thus, the regulation of PAPP-A production by injury in skeletal muscle needs to be confirmed by Western immunoblot analysis once antibodies against rodent PAPP-A become available. Although our data indicate that PAPP-A may be an important determinant in skeletal muscle repair in response to injury, the relation of the enhanced PAPP-A expression to skeletal muscle regeneration requires a thorough investigation in future studies.


Figure 1
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  		FIG. 1. PAPP-A expression increased after muscle injury. Skeletal muscle damage was created by physically crushing the TA muscle (see Materials and Methods). Total RNAs were isolated from injured (right leg) and control (Cont) TA (left leg) muscles 3, 5, and 14 d after surgery. mPAPP-A was quantitated by real-time PCR using primers specific for mouse PAPP-A (Table 1Go). PAPP-A mRNA levels were adjusted for GAPDH expression. Endogenous PAPP-A mRNA levels increased significantly 5 d after TA muscle injury (n = 3 for d 3 and 5; n = 6 for d 14). *, P < 0.05 and **, P < 0.01 vs. control uninjured TA.

 
Generation of skeletal muscle-specific PAPP-A transgenic mice
To investigate the role of PAPP-A in skeletal muscle in vivo, we generated transgenic mice which overexpress hPAPP-A in skeletal muscle using the human skeletal {alpha}-actin promoter. The design of the transgenic construct is shown in Fig. 2AGo. Founders were identified by PCR analysis of tail DNA (Fig. 2BGo). Recombinant PAPP-A was detected in the CM of cultured skeletal muscle from F1 generation transgenic mice of each line (Fig. 2CGo). Accordingly, IGFBP-4 proteolysis (Fig. 2DGo, upper gel) was significantly increased in the transgenic muscle CM, compared with the wild-type muscle CM, in all three lines. The observed increase in IGFBP-4 proteolysis was not contributed by other proteases because addition of PAPP-A neutralization antibody to transgenic mouse CM completely abolished proteolysis of IGFBP-4 (Fig. 2DGo, lower gel).


Figure 2
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  		FIG. 2. Development of skeletal muscle-specific PAPP-A transgenic mice. A, Design of PAPP-A transgenic construct. B, Founders were identified by PCR analysis of the tail DNA using a pair of primers specific for hPAPP-A cDNA (Table 1Go). PC, PAPP-A plasmid control. C, CM of cultured muscle isolated from F1 generation mice was prepared and subjected to immunoblot analysis with hPAPP-A antibody (see Materials and Methods). D, Upper gel, proteolysis of IGFBP-4 was evaluated in the presence of transgenic (Tg.) muscle CM or wild-type (Wt.) muscle CM (see Materials and Methods). D, Lower gel, IGFBP-4 proteolysis by Tg muscle CM was evaluated in the presence of control IgG or anti-PAPP-A IgG followed by IGF-II ligand blot analysis (see Materials and Methods). Addition of PAPP-A neutralization IgG abolished Tg muscle CM-induced IGFBP-4 proteolysis. E, Free IGF-I concentrations in the CM were measured 2 h after adding fresh DMEM/10% FCS after the initial incubation of 43 h (see Materials and Methods).

 
Consistent with the increased IGFBP proteolysis activity, free IGF-I concentration in the transgenic muscle CM was significantly increased (Fig. 2EGo). Free IGF-I concentrations were not proportional to the amount of PAPP-A in the CM of muscles isolated from different lines because the amount of IGFBPs, not the amount of the PAPP-A, is the limiting factor under these conditions.

PAPP-A transgenic mice exhibit increased somatic growth and skeletal muscle mass
Because skeletal muscle contributes to more than 40% of the body weight, we would expect an increase in body weight if PAPP-A significantly increases muscle mass. Compared with the wild-type littermates, female transgenic mice from all three lines showed a 20–50% increase in body weight by 10 wk of age (Fig. 3AGo). The body weight of two male transgenic mice from founder 167 also showed a higher body weight, compared with that of a wild-type littermate (no transgenic males were found in the litters of founder 154 and 156). These results suggest that the PAPP-A-enhanced postnatal growth is gender independent. The average body weight of female transgenic mice pooled from all three lines was significantly higher than the average body weight of the pooled wild-type littermates at both 10 wk and 6 months of age (Fig. 3BGo). Consistently, the length of the body (nose to tail tip) was increased by 9.1% in transgenic mice at an age of 6 months (P < 0.05) (data not shown).


Figure 3
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  		FIG. 3. PAPP-A transgenic mice exhibited increased body weight and skeletal muscle mass. A, Body weight of F1 generation mice from a litter of each transgenic (Tg) line was recorded at age of 10 wk. B, Average body weight was significantly higher in female F1 generation mice pooled from all three lines. C, One-day-old F3 generation newborn pups [six Tg and seven wild-type (Wt)] were weighed, and their genotypes were determined by immunoblot analysis of the cultured muscle CM. D, The forearm CSA, determined by pQCT on 10-wk-old live female F1 offspring pooled from three lines, was significantly increased in transgenic mice. *, P < 0.05; **, P < 0.01 vs. Wt.

 
To determine whether PAPP-A overexpression affects prenatal growth, newborn F3 generation pups (1 d old) were weighed and their genotypes were determined by immunoblot analysis of the CM of cultured muscle. The transgenic pups showed a significantly higher birth weight than their wild-type littermates (~12%, P < 0.05, Fig. 3CGo). These data suggest that PAPP-A overexpresion was able to enhance not only postnatal but also prenatal growth.

The CSA of the forearm (mainly determined by muscle mass) measured by pQCT on 10-wk-old live animals was increased by approximately 50% (P < 0.05) in transgenic mice, compared with littermate control mice (Fig. 3CGo). This increase in skeletal muscle mass was further confirmed by histological examination of midbelly sections of the TA muscles (Fig. 4Go, A and B). Transgenic mice showed a significant increase in the TA muscle wet weight (P < 0.001, Fig. 4CGo) and midbelly CSA (P < 0.05, Fig. 4DGo), compared with their wild-type littermates. Additionally, TA muscle fiber CSA was significantly larger in transgenic mice (Fig. 4EGo). Taken together, these data suggest that PAPP-A overexpression causes skeletal muscle hypertrophy.


Figure 4
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  		FIG. 4. PAPP-A transgenic mice exhibited increased TA muscle size and myofiber CSA. Data were derived from 6-month-old female F1 offspring from three different transgenic lines [two transgenic (Tg) and one wild-type (Wt) mouse from line 154; one Tg mouse and one Wt mouse from line 156; one Tg mouse and two Wt mice from line 167]. TA muscle was weighed and fixed in 10% formalin. Five micromole cross-sections were subjected to H & E staining or laminin staining. A, Representative digital images (H & E staining) of TA muscle cross-sections. B, Magnified (x20) laminin staining of midbelly section of TA muscle. C, Average wet weight of TA muscle. D, Average CSA of midbelly section of TA muscle. E, Average TA muscle myofiber CSA. *, P < 0.05; ***, P < 0.001 vs. Wt.

 
The anabolic effects of PAPP-A overexpression were confirmed by analysis of the F2 generation progeny (60 d old) derived from line 167. Analysis of the CM from ex vivo cultured TA muscle confirmed production of hPAPP-A and increased IGFBP-4 proteolysis (data not shown). The body weight and lean body mass measured by DEXA, and the TA muscle myofiber area were all significantly increased in transgenic mice, compared with the littermate control (Fig. 5Go). Either lean mass or body fat per gram of body weight was not significantly different between transgenic mice and control mice (data not shown). The increase in muscle size was also reflected by an increase in the CSA of the midbelly of the forearm in a representative pair of mice (Fig. 5Go, inset). Because an increase in heart and kidney weight was also observed in these PAPP-A transgenic mice, we performed an additional experiment to examine whether the size of other tissues/organs were also affected by PAPP-A overexpression. A moderate increase in the weight of the collected tissues/organs was observed in PAPP-A transgenic mice, although the magnitude of the increase for each organ varied (Table 2Go).


Figure 5
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  		FIG. 5. Phenotypic analysis of F2 generation progeny from PAPP-A transgenic line 167. F2 generation pups were obtained by breeding an F1 male transgenic mouse from line 167 with B6 control female mice. At 60 d of age, mice [three wild-type (Wt) and four transgenic (Tg) pooled from two litters] were subjected to DEXA analysis for lean body mass and fat mass (see Materials and Methods). Myofiber area was determined as described (see Materials and Methods). Inset, Frozen forearms from a representative Wt. and Tg. pair were cut at the midbelly (the thickest point of the forearm) and stained with Coomassie blue dye and photographed. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. Wt.

 

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  		TABLE 2. Body and organ weight of PAPP-A transgenic mice and wild-type littermates (grams)

 
To explain the mechanism for the increase in the weight of nonskeletal muscle organs in PAPP-A transgenic mice, we compared the hPAPP-A transgene expression in skeletal muscle vs. several other tissues/organs using RT-PCR analysis. The hPAPP-A transgene was highly expressed in transgenic skeletal muscle as expected (Fig. 6AGo). A low level of PAPP-A transgene expression was detected in heart, brain, and spleen (Fig. 6AGo). Expression of PAPP-A transgene in other remaining organs is negligible.


Figure 6
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  		FIG. 6. Analysis of hPAPP-A transgene expression in tissues and hPAPP-A activity in serum. Tissues and serum were collected from F2 progeny of line 167 [three wild-type (Wt) and four transgenic (Tg) mice]. A, Total RNA was isolated from various tissues from individual mouse. Level of mRNA for each gene was evaluated by RT-PCR. The hPAPP-A transgene was highly expressed in skeletal muscle and weakly expressed in heart, brain, and spleen. B, Eight microliters of serum from each individual mouse were subjected to Western immunoblot analysis using an antibody specific for hPAPP-A. C, Fifty nanograms IGFBP-4 were incubated for 5 h at 37 C with 1.5 µl serum in the presence of 25 ng IGF-II and subjected to IGF-II ligand blot analysis.

 
Because PAPP-A is a secreted protein, we examined whether skeletal muscle-derived hPAPP-A entered circulation. Immunoblot analysis, using a hPAPP-A-specific antibody, revealed the presence of significant amounts of hPAPP-A in the serum of all individual transgenic mice but not the serum of their littermate control mice (Fig. 6BGo). Consistent with the increased levels of PAPP-A, IGFBP-4 proteolysis was dramatically increased in the presence of the serum from transgenic mice, compared with that of wild-type mice (Fig. 6CGo).

Ex vivo evidence that PAPP-A enhances skeletal myogenesis via increasing local IGF bioavailability
We also examined whether transgenic overexpression of hPAPP-A has any effect on endogenous PAPP-A or IGF-I production in skeletal muscle. As shown in Fig. 7Go, overexpression of hPAPP-A in the skeletal muscle of mice did not affect the expression of either endogenous PAPP-A or IGF-1. Interestingly, transgenic mice showed a significantly lower serum total IGF-I concentration, compared with that of wild-type littermates (Fig. 8AGo). Because no reliable methods for quantitation of rodent-free IGF-I or IGF-II are available, we spiked mouse serum with human IGF-I and then determined the concentration of free hIGF-I in the serum using the human IGF-I ELISA. No significant difference in free hIGF-I was observed between transgenic serum and wild-type serum (data not shown). To further confirm that IGF bioavailability in circulation was not affected by PAPP-A overexpression, we evaluated the IGF bioactivity in the serum using the cell proliferation assay. Human osteosarcoma MG63 cells were used in this assay because they do not produce any detectable levels of PAPP-A and are very responsive to IGF-I treatment. We found that cell number was significantly increased in the presence of 0.5% heat-inactivated transgenic mouse serum or wild-type mouse serum (Fig. 8BGo). However, no significant difference in cell number was observed in cultures treated with transgenic vs. wild-type mouse serum. Neutralization of free IGFs by addition of PAPP-A-resistant IGFBP-4 (PRBP-4) failed to reduce cell proliferation induced by either serum. These results suggest that overexpression of PAPP-A in muscle is unlikely to significantly affect IGF bioavailability in circulation, even though the muscle-produced PAPP-A was leaked into circulation. However, this conclusion needs to be confirmed in future studies after reliable mouse-free IGF-I assays become available.


Figure 7
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  		FIG. 7. Human PAPP-A overexpression in skeletal muscle did not alter expression of endogenous PAPP-A or IGF-I. Total RNAs isolated from various tissues (Fig. 6Go) were used to evaluate expression of endogenous PAPP-A [mouse PAPP-A (mPAPP-A)] and IGF-I [mouse IGF-I (mIGF-I)] (see Materials and Methods).

 

Figure 8
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  		FIG. 8. Effect of PAPP-A overexpression on serum total IGF-I concentration and serum IGF bioactivity. A, Serum total IGF-I concentrations were measured by IGF-I RIA [six wild-type (Wt.) serum samples and eight transgenic (Tg.) serum samples, 10-wk-old F3 generation mice]. B, Human osteosarcoma MG63 cells were seeded in 24-well plates (30,000 cells/well) in DMEM/10%FCS. After a 20-h incubation, cells were starved in serum-free medium for 10 h. Then cells were cultured in DMEM/0.1% BSA supplemented with 65 C heat-inactivated Tg. or Wt. serum (S) at final concentration of 0.5% in the presence or absence of PRBP-4 (400 ng/ml). After a 40-h incubation, cell number was determined as described in Materials and Methods (n = 4). Note: the activity of PAPP-A can be inactivated at 65 C for 30 min, whereas IGF-I remains biologically active after this treatment (Qin, X., and S. Mohan, unpublished data). N.S., Not significant (P > 0.05).

 
To determine whether the enhanced skeletal muscle mass of transgenic mice is due to increased IGF bioavailability in muscle tissue, we compared the myogenic activity of the CM from ex vivo cultured muscle isolated from transgenic vs. littermate control mice, in the presence or absence of PRBP-4. The proliferation of C2C12 myoblasts was significantly increased on addition of CM from the cultured muscle of transgenic mice, compared with that of littermate control mice. This enhanced proliferative effect induced by the transgenic muscle CM was abolished in the presence of PRBP-4 peptide (Fig. 9AGo). Consistent with these results, the PAPP-A-induced increase in free IGF-I concentration did not occur in the presence of PRBP-4 (Fig. 9BGo). These data indicate that the increased proliferation of C2C12 myoblasts is due to the increased bioavailability of IGF-I in the presence of transgenic muscle CM.


Figure 9
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  		FIG. 9. Proliferation of C2C12 myoblasts treated with CM collected from ex vivo cultured transgenic vs. control muscle in the presence or absence of IGF inhibitor. C2C12 myoblasts were seeded in 24-well plates and cultured in muscle CM (Fig. 2Go) diluted three times with DMEM/10% FCS. PRBP-4 was added to a final concentration of 500 ng/ml when indicated. After a 48-h culture, cell number was determined using AlamarBlue dye (A), and free IGF-I concentration (B) was determined by ELISA. The number of myoblasts was significantly increased in cultures treated with the CM of cultured transgenic muscle, compared with CM of control muscle, and this increase was abolished in the presence of PRBP-4 peptide.

 
Protease domain in PAPP-A is indispensable to its biological activity in myogenic cells
To provide additional evidence that PAPP-A acts to enhance myogenesis via cleaving IGFBPs, consequently increasing IGF bioavailability, we prepared a mutant PAPP-A containing a point mutation in the Zn finger motif (E483/A). We then compared the effect of this protease-inactive PAPP-A (E483/A) vs. the wild-type PAPP-A on myoblast proliferation and differentiation. Sequencing of the PAPP-A (E483/A) plasmid confirmed the presence of the desired E483/A point mutation (Fig. 10AGo) and absence of any unwanted mutations in the PAPP-A cDNA sequence (data not shown). Immunoblot analysis of the CM from transfected C2C12 myoblasts revealed the presence of the recombinant PAPP-A (E483/A) protein, which comigrated with the wild-type PAPP-A (Fig. 10Go, B and C). CM containing PAPP-A (E483/A) failed to cleave any of the three PAPP-A substrate IGFBPs (Fig. 10DGo).


Figure 10
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  		FIG. 10. Preparation and characterization of the protease-defective PAPP-A mutant PAPP-A (E483/A). A, Presence of the E483/A mutation was confirmed by DNA sequencing of the noncoding strand of the PAPP-A (E483/A)/pFLAG plasmid. B, C2C12 myoblasts were transfected with 2 µg/well of pFLAG vector DNA (Vector) or PAPP-A/pFLAG DNA (Wt.) or PAPP-A (E483/A)/pFLAG DNA (E483/A). CM was collected after a 48-h incubation. Forty microliters of CM were subjected to immunoblot analysis with the FLAG antibody. C, Forty microliters of CM were subjected to immunoblot analysis with the hPAPP-A antibody. D, A mix of 60 ng IGFBP-2 or IGFBP-4 or IGFBP-5 was incubated with 1.5 µl of CM in the presence of 25 ng IGF-II for 4 h at 37 C and then subjected to 125I-IGF-II ligand blot analysis. In contrast to Wt. PAPP-A, the mutant PAPP-A (E483/A) did not cleave IGFBP-2 or IGFBP-4 or IGFBP-5.

 
Additionally, we determined whether transient overexpression of PAPP-A (E483/A) could stimulate C2C12 myoblast proliferation and differentiation. Expression of mutant PAPP-A and wild-type PAPP-A protein was confirmed by immunoblot analysis on the CM from transfected C2C12 cells using a hPAPP-A antibody (Fig. 11AGo). Free IGF-I was not detectable in the CM from myoblasts transfected with either vector plasmid or PAPP-A (E483/A) plasmid, whereas free IGF-I concentration increased to approximately 5 ng/ml (Fig. 11BGo) in the CM from myoblasts overexpressing wild-type PAPP-A. Overexpression of wild-type PAPP-A increased C2C12 number (Fig. 11CGo) and CK activity, a biochemical marker for myoblast differentiation (Fig. 11DGo). In contrast, overexpression of the mutant PAPP-A had no effect on any of these parameters.


Figure 11
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  		FIG. 11. Transient overexpression of PAPP-A (E483/A) in C2C12 myoblasts did not increase IGF-I bioavailability or myoblast proliferation/differentiation. C2C12 myoblasts were seeded in six-well plates and transfected with indicated amount of vector plasmid or PAPP-A (E483/A) plasmid (E483/A) or the wild-type PAPP-A plasmid (WT). Forty-eight hours after transfection, CM was collected for evaluation of PAPP-A production (A) and free IGF-I measurement (B). Immediately after collection of CM, cell number was determined by incubating live cells with AlamarBlue dye for 1 h (C). After removing the dye, cells were cultured in the DMEM/2% horse serum for an additional 72 h, and CK activity in cell lysate (10 µg protein) was measured (D).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Past studies have provided unambiguous evidence that IGFs are indispensable to skeletal muscle development, maintenance, and regeneration (10, 11). The central role of IGF-I in the regulation of skeletal muscle formation is strongly supported by genetic studies demonstrating that IGF-I or IGF-I receptor knockout in mice causes severe muscle atrophy (33, 34), whereas overexpression of IGF-I in skeletal muscle leads to skeletal muscle hypertrophy (35, 36, 37). Whereas the myogenic action of IGFs is well established, little information is available regarding the physiological significance of IGFBPs in regulating the local action of IGFs in muscle. In this study we found that expression of PAPP-A was increased in skeletal muscle after muscle crush injury (Fig. 1Go). It is known that skeletal muscle produces IGFBP-3, -4, -5, and -6, with IGFBP-3 and -5 being the most abundant (36, 38). Thus, an increase in PAPP-A production after muscle injury could lead to an increase in the local concentration of free IGF, which would in turn promote proliferation and subsequent fusion of myoblasts/satellite cells into regenerating myofibers. This contention needs to be confirmed in future studies using animal models in which the PAPP-A gene or activity in muscle is inactivated.

An important finding from this study is that overexpression of PAPP-A in skeletal muscle significantly increased skeletal muscle mass and myofiber CSA (Figs. 3–5GoGoGo). These anabolic effects are manifested between 6 and 10 wk of age and are maintained during aging. The muscle phenotype in PAPP-A transgenic mice is similar to that observed in transgenic mice overexpressing various IGF-I isoforms, using muscle-specific promoters (11, 35, 36, 37). Thus far, our findings do not support a major role for circulating IGFs in the regulation of skeletal muscle formation in PAPP-A transgenic mice. First, serum total IGF-I concentration was not increased but rather decreased in transgenic mice (Fig. 8Go). This phenotype is similar to that observed in IGFBP-3/-4,/-5 triple knockout mice (39). Although serum-free IGF-I concentration was not measured, due to lack of a rodent-free IGF-I assay, myoblast proliferation studies did not provide solid evidence that IGF bioavailability is altered in the blood of transgenic mice (Fig. 8Go). In contrast, IGF bioavailability is dramatically increased in ex vivo cultured transgenic muscles (Fig. 2DGo). Proliferation of C2C12 myoblasts was significantly increased in the presence of transgenic muscle CM than in the presence of wild-type muscle CM. The increased mitogenic activity in the PAPP-A transgenic muscle CM results from increased IGF bioavailability because enhanced myoblast proliferation is abolished in the presence of noncleavable IGFBP-4 peptide, which neutralizes free IGFs (Fig. 9Go). Thus, these ex vivo data suggest that the increased skeletal myogenesis in PAPP-A transgenic mice is most likely caused by an increase in IGF bioavailability in the muscle tissue local environment.

Evidence to support an IGF-dependent mechanism for PAPP-A’s action in muscle is also derived from our structure-function analysis of PAPP-A. Human PAPP-A is one of the largest known secreted proteins (a homodimer of 1547 amino acids). It contains multiple functional domains and interacts with several biologically active proteins such as proMBP, complement 3, and heparin (24, 25, 40, 41). Besides acting as a proteolytic enzyme, it is unknown whether PAPP-A has other biological functions through the action of other functional domains and/or interacting with other proteins. In this study we prepared a mutant PAPP-A (E483/A) that contains a point mutation in its Zn finger motif. Consistent with a previous report, this mutant was unable to cleave IGFBP-4 (42). We also found this mutant PAPP-A unable to cleave IGFBP-2 and IGFBP-5 (Fig. 10DGo), an important feature not previously reported by other investigators (42). Importantly, this mutant PAPP-A had no effect on IGF bioavailability or myoblast proliferation/differentiation (Fig. 11Go).

Although our in vitro and ex vivo data suggest that PAPP-A may act to increase muscle mass through increasing IGF bioavailability in muscle tissue, existence of other IGF-independent mechanisms in vivo cannot be completely excluded at this time. In this regard, it has recently been reported that IGFBP-3/-4/-5 triple knockout mice did not exhibit an increase but rather a decease in serum total IGF-I level, serum IGF bioactivity, and somatic growth as well as skeletal muscle mass (39). Based on the level of PAPP-A produced in the muscle of PAPP-A transgenic mice, intact IGFBP-2, -4, and -5 are not expected to be present in the interstitial fluid of muscles. Despite some similarities in these two mouse models, opposite phenotypes (except for a decrease in serum total IGF-I concentrations) were seen between PAPP-A transgenic mice and IGFBP-3/-4/-5 triple knockout mice. Thus, future studies are needed to explain these discrepancies.

Because PAPP-A is a secreted protease, it may exert its anabolic effects in both a paracrine and endocrine manner, as with the case of IGFs. PAPP-A transgenic mice show a significant increase in prenatal and postnatal growth (Fig. 3Go). This phenotype is opposite to that observed in PAPP-A knockout mice (43) and similar to that observed in transgenic mice, which overexpress IGF-I Ea isoform using the myosin light chain promoter (37). The increase in body weight in PAPP-A transgenic mice is due to an increase not only in skeletal muscle mass but also other organs as well. A moderate increase in the weight of several nonskeletal muscle organs was observed in PAPP-A transgenic mice, although the magnitude of increase in each organ varied (Fig. 5AGo and Table 2Go). These generalized effects are unlikely to be caused by an alteration of IGF bioavailability in circulation. Despite the release of PAPP-A into the blood, serum total IGF-I concentration or serum IGF bioactivity is not increased in PAPP-A transgenic mice (Fig. 8Go). Several possibilities may explain the global effect of PAPP-A overexpression on somatic growth. First, weak expression of the PAPP-A transgene in nonskeletal muscle organs/tissues may lead to a significant increase in IGF bioavailability locally in these tissues/organs. In this regard, increased heart weight is consistent with expression of the PAPP-A transgene mRNA in this organ. It is reported that cardiac hypertrophy occurs in avian skeletal {alpha}-actin/IGF-I transgenic mice that overexpressed IGF-I transgene in the heart (35). Interestingly, PAPP-A overexpression had very little effect on brain weight, whereas a significant amount of PAPP-A transgene expression was detected in this tissue. It is possible that brain volume is determined by skull size, which is not increased in PAPP-A transgenic mice. Alternatively, an unknown mechanism may exist in the brain to antagonize the effect of PAPP-A on this tissue. Second, although PAPP-A is an extremely large protein (>400 kDa), detection of a significant amount of hPAPP-A in the blood of transgenic mice clearly indicates that PAPP-A can penetrate the endothelial barrier of the microblood vessels. It is very likely that PAPP-A can be released from circulation to act on other tissues/organs, thereby increasing local IGF bioavailability. The potential effect of circulating PAPP-A on overall somatic growth is consistent with our finding that bone-specific PAPP-A transgenic mice did not show an increase in body weight (32) because hPAPP-A was not detected in the blood (data not shown).

In summary, our studies demonstrate that targeted hPAPP-A overexpression in skeletal muscle significantly increased prenatal/postnatal growth and skeletal myogenesis in mice. Our in vivo and ex vivo data support the hypothesis that an increase in local IGF bioavailability is the primary, if not the only, cause for the PAPP-A-enhanced myogenesis observed in vivo. These studies also provide first direct experimental evidence that increasing the bioavailability of endogenous IGFs through enhancing IGFBP proteolysis can lead to similar, if not greater, anabolic responses, compared with increasing IGF-I production.


   Acknowledgments
 
We thank Nancy Lowen and Carolyn Hargrave for performing histochemical analysis, Heather Davidson for assistance with pQCT analysis, Yan Hu for DNA sequencing, and Dr. Mehran Amoui and Dr. Kesavan Chandera for assistance with the real-time PCR procedure. All work was performed at facilities provided by the Jerry L. Pettis Memorial Veterans Affairs Medical Center (Loma Linda, CA).


   Footnotes
 
This work was supported by a grant from the Department of Veterans Affairs (Merit Review to X.Q.).

Current address for A.K.: Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, 500 South Preston Street, Louisville, Kentucky 40292.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 27, 2007

Abbreviations: CK, Creatine kinase; CSA, cross-sectional area; CM, conditioned medium; DEXA, dual energy X-ray absorptiometry; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, human; H & E, hematoxylin and eosin; IGFBP, IGF binding protein; PAPP-A, pregnancy-associated plasma protein; PBS, phosphate buffered saline; pQCT, peripheral quantitative computed tomography; PRBP-4, PAPP-A-resistant IGFBP-4; proMBP, proform of eosinophil major basic protein; TA, tibialus anterior.

Received March 5, 2007.

Accepted for publication September 17, 2007.


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Top
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 Introduction
 Materials and Methods
 Results
 Discussion
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