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J Appl Physiol 101: 183-188, 2006; doi:10.1152/japplphysiol.00636.2005
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Expression of insulin-like growth factor I, insulin-like growth factor binding proteins, and collagen mRNA in mechanically loaded plantaris tendon

Jens L. Olesen,1 Katja M. Heinemeier,1 Fadia Haddad,2 Henning Langberg,1 Allan Flyvbjerg,3 Michael Kjær,1 and Kenneth M. Baldwin2

1Institute of Sports Medicine, Bispebjerg Hospital, Copenhagen, Denmark; 2Department of Physiology and Biophysics, University of California, Irvine; and 3Medical Research Laboratories, Aarhus University Hospital, Aarhus, Denmark

Submitted 28 May 2005 ; accepted in final form 24 March 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Insulin-like growth factor I (IGF-I) is known to exert an anabolic effect on tendon fibroblast production of collagen. IGF-I's regulation is complex and involves six different IGF binding proteins (IGFBPs). Of these, IGFBP-4 and -5 could potentially influence the effect of IGF-I in the tendon because they both are produced in fibroblast; however, the response of IGFBP-4 and -5 to mechanical loading and their role in IGF-I regulation in tendinous tissue are unknown. A splice variant of IGF-I, mechano-growth factor (MGF) is upregulated and known to be important for adaptation in loaded muscle. However, it is not known whether MGF is expressed and upregulated in mechanically loaded tendon. This study examined the effect of mechanical load on tendon collagen mRNA in relation to changes in the IGF-I systems mRNA expression. Data were collected at 2, 4, 8 and 16 days after surgical removal of synergistic muscle to the plantaris muscle of the rat, thus increasing the load to plantaris muscle and tendon. Nearly a doubling of the tendon mass was observed after 16 days of loading. A rapid rise in tendon procollagen III mRNA was seen after 2 days whereas the increase in procollagen I mRNA was significant from day 8. MGF was expressed and upregulated in loaded tendon tissue with a faster response than IGF-I, which was increased from day 8. Finally, IGFBP-4 mRNA was increased with a time pattern similar to procollagen III, whereas IGFBP-5 decreased at day 8. In conclusion, loading of tendon tissue results in an upregulation of IGF-I, IGFBP-4, and procollagen and is associated with an increase in tendon mass. Also, MGF is expressed with an early upregulation in loaded tendon tissue. We suggest that the IGF-I system could be involved in collagen synthesis in tendon in response to mechanical loading.

insulin-like growth factor I; mechano-growth factor; collagen; tendon; mechanical loading; insulin-like growth factor binding proteins

LOADING OF SKELETAL MUSCLE is known to induce hypertrophy with both qualitative and quantitative remodeling of muscle proteins (5, 17, 40). However, less well described is the scaffold that supports the contractile muscle proteins in transforming their contraction to a functional force, i.e., the connective tissue including tendon tissue. Previous studies have demonstrated that loading gives rise to an enlargement of tendon tissue and to an increase in the collagen synthesis both at protein and mRNA levels (2, 32, 36). The response of tendon to a change in the loading pattern of the muscle-tendon unit is therefore of interest because changes in load can lead to an inadequate response from the tendon, resulting in overloading and subsequent injury (3, 27, 31). It is important to have knowledge of factors that regulate the adaptation of collagen synthesis in tendon during changes in loading, because this gives an understanding of the pathogenesis of the overuse injuries in tendons and thus helps to evaluate the implication of a treatment. Several cytokines and growth factors, e.g., insulin-like growth factor I (IGF-I), transforming growth factor-beta, and interleukin-6, have been implicated in mediating the effect of increased loading of the fibroblasts in the tendon to produce collagen (7, 16, 23, 29, 41). Of these, IGF-I may play a key role given its known role as a multipotent stimulator in various tissues, e.g., bone and skeletal muscle (1, 15, 33, 34).

Two splice variants of the IGF-I gene are expressed in rodent skeletal muscle, namely IGF-I Ea and IGF-I Eb. The IGF-I Eb isoform, known as mechano-growth factor (MGF), is upregulated in exercised and damaged muscle. This isoform is believed to be important for local tissue repair and adaptation (18, 45). However, it is not known whether MGF is expressed and influenced by mechanical loading in tendon tissue.

In addition to IGF-I, IGF binding proteins (IGFBPs) are known to interact with IGF-I and have independent functions (13, 43). In the overloaded muscle an increased expression of IGFBP-4 mRNA has been shown to occur, whereas IGFBP-5 expression was either unchanged or decreased (4, 20). Both IGFBP-4 and IGFBP-5 are produced by fibroblasts, but it is unknown how these factors respond to mechanical loading of the tendon (11).

We therefore hypothesized that, in response to an increased loading state, stimulated expression of collagen mRNA in the tendon will be associated with increases in IGF-I and IGFBP-4 mRNA, similar to that observed in the muscle (20, 22, 30). For IGFBP-5 mRNA a constant level or even a decrease in expression is expected. Furthermore, we proposed that MGF is not solely an IGF-I isoform found in skeletal muscle but that it could also be expressed in mechanically loaded tendon tissue.

The aim of this study was to examine collagen mRNA in the tendon of the plantaris muscle and relate this pattern to the time course of change in IGF-I, MGF, and IGFBP mRNA expression in response to increased load and thereby to gain further knowledge about the tendon's molecular adaptation to changes in loading. Expression patterns were examined at 2, 4, 8, and 16 days after induction of a known loading model of the plantaris muscle and tendon in rodents (6).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Experimental design.   Forty-eight young adult female Sprague-Dawley rats weighing 245 ± 8 g were randomly assigned to either a control (n = 16) or a loaded plantaris group (n = 32). Animals in the loaded group subsequently were assigned randomly to four subgroups with eight rats in each and studied on days 2, 4, 8, and 16 after the surgical procedure. Rats subjected to surgical manipulation were anesthetized with a cocktail consisting of ketamine hydrochloride (50 mg/kg), xylazine (4 mg/kg), and acepromazine (1 mg/kg) intramuscularly. The plantaris muscle in one leg of the rat was functionally loaded by surgical removal of its synergists consisting of both the gastrocnemius and the soleus together with part of the Achilles tendon (5). Sham operations were performed on the contralateral legs with skin incisions and presentation of the individual muscle groups. All animals were housed in standard cages and allowed food and water ad libitum. The institutional animal research committee approved the treatment protocol.

Tissue processing.   At each time point, animals from appropriate subgroups were weighed and killed via an overdose of Pentosol euthanasia solution (Med-Pharmex). At the cessation of heartbeat, a skin incision was made, and the plantaris muscle in situ with tendon was isolated. A fast dissection of the muscle-tendon unit was performed into three macroscopic parts: distal tendon, muscle belly, and the myotendinous junction. The two latter parts were used for other analytical purposes. All samples were immediately snap-frozen, weighed, and stored at –80°C for subsequent analyses. To limit variability in collecting the tendon tissue, and to be consistent across all groups, only one person was in charge of all the dissection of the tendon using a magnifying glass to better observe the anatomical mark of the tendon, myotendon, and muscle.

Total RNA isolation.   Total RNA was extracted from preweighed frozen tendon samples by using the TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol, which is based on the method described by Chomczynski (12). For sufficient material, the control samples were pooled in groups of four and the loaded tendons samples were pooled in pairs. Extracted RNA was precipitated from the aqueous phase with ethanol, dried, and suspended in a known volume of nuclease-free water. The RNA concentration was determined by optical density at 260 nm (OD260; using an equivalent of 40 µg RNA/ml per unit OD260). The tendon total RNA concentration was calculated on the basis of total RNA yield and the weight of the analyzed sample. The RNA samples were stored frozen at –80°C until subsequent use in semiquantitative RT-PCR procedures. RNA quality was determined on the basis of ribosomal RNA pattern observed after gel electrophoresis of ~0.5 µg of RNA on 1% agarose gel and ethidium bromide staining. A good RNA sample contained both 28S, 18S, and 5S ribosomal RNA bands. In contrast, degraded sample does not contain or has very little 28S compared with 18S and 5S. Only integer RNA samples were used for the RT-PCR analyses.

RT.   One microgram of total RNA was reverse transcribed for each tendon sample by using the SuperScript II RT from Invitrogen and a mix of oligo(dT) (100 ng/reaction) and random primers (200 ng/reaction) in a 20-µl total reaction volume at 45°C for 50 min, according to the provided protocol. At the end of the RT reaction, the tubes were heated at 90°C for 5 min to stop the reaction and then the samples were stored at –80°C until used in the PCR reactions for specific mRNA analyses (see paragraph below).

PCR.   A semiquantitative RT-PCR method using 18S as an internal standard (Ambion, Austin, TX) was applied to study the expression of specific mRNAs of interest, including IGF-I, MGF, IGFBP-4, IGFBP-5, procollagen type I alpha 2, and procollagen type III alpha 1. The IGF-I primer used for this study detects all IGF-I splice variants. The MGF variant appears as a separate band, which is not integrated when the densitometry measurements are made. The sequence for the primers used for the specific target mRNAs is shown in Table 1. These primers were purchased from Qiagen. In each PCR reaction, 18S ribosomal RNA was coamplified with the target cDNA (mRNA) to serve as an internal standard and to allow correction for any differences in starting amounts of total RNA.


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  		Table 1. Primer sequences

 
For the 18S amplification, we used the Alternate 18S Internal Standards (Ambion), which yields a 324-bp product. The 18S primers were mixed with competimers at an optimized ratio that could range from 1:6 to 1:12, depending on the abundance of the target mRNA. Inclusion of 18S competimers was necessary to bring down the 18S signal, which allows its linear amplification to the same range as the coamplified target mRNA (semiquantitative RT-PCR kit protocol, Ambion, Austin, TX).

For each target mRNA, the RT and PCR reactions were carried out under identical conditions by using the same reagent premix for all the samples to be compared in the study. To validate the consistency of the analysis procedures, at least one representative from each group was included in each RT-PCR run.

One microliter of each RT reaction (0- to 30-fold dilution depending on target mRNA abundance) was used for the PCR amplification. The PCR reactions were carried out in the presence of 2 mM MgCl2 by using standard PCR buffer (GIBCO, Carlsbad, CA), 0.2 mM dNTP, 1 µM specific primer set, 0.5 µM 18S primer-competimer mix, and 0.75 unit of Taq DNA polymerase (GIBCO) in 25 µl total volume. Amplifications were carried out in a Stratagene Robocycler with an initial denaturing step of 3 min at 96°C, followed by 23–27 cycles of 1 min at 96°C, 1 min at 55°C (52–60°C depending on primers), 1 min at 72°C, and a final step of 3 min at 72°C. PCR products were separated on a 2% agarose gel by electrophoresis and stained with ethidium bromide, and signal quantification was conducted by digitized image analyses. The image of the UV-fluorescent gel was captured with a digital camera, followed by digitization and analyses using Image Quant Software (Molecular Dynamics). In this approach, each specific mRNA signal is normalized to its corresponding 18S. For each primer set, PCR conditions (cDNA dilutions, 18S competimer-primer mix, MgCl2 concentration, and annealing temperature) were set to optimal conditions so that both the target mRNA and 18S product yields were in the linear range of the semilog plot when the yield is expressed as a function of the number of cycles.

Statistical analysis.   All values are reported as means ± SD. Each data set was analyzed for deviations from Gaussian distribution using D'Agostino and Pearson omnibus normality test. For each time point, treatment effect was determined by ANOVA with Newman-Keuls post hoc testing by using the Prism software package (Graphpad, San Diego, CA). For all statistical tests, the 0.05 level of confidence was accepted for statistical significance.


   RESULTS
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 METHODS
 RESULTS
DISCUSSION
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 ACKNOWLEDGMENTS
 REFERENCES
 
As illustrated in Fig. 1, both plantaris muscle and tendon of the rat underwent a gain in wet weight after the removal of synergist muscles without any significant increase in total body weight. For the muscle, the increase was significant throughout the experiment time course with an elevation of ~70%. For tendon, the increase was somewhat slower and reached significance on day 16 whereby a 100% increase compared with the control was observed.


Figure 1
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  		Fig. 1. Wet weight changes of plantaris muscle (solid bars) and plantaris tendon (open bars) during 16 days of loading by surgical removal of the gastrocnemius and soleus muscle. Data are means ± SD of n = 8 or 16 (control)/group at each time point. *P < 0.05 vs. control.

 
Tendon IGF-I system.   Our results show that IGF-I mRNA levels relative to 18S increased significantly at day 8 of plantaris loading and remain elevated at day 16. This response appears to precede the elevation in tendon wet weight (Fig. 2A). With regard to MGF, the IGF-I isoform related to adaptation in muscle, its mRNA levels were measurable in control tendon tissue where they were observed to increase significantly by day 4 and remained elevated through to day 16 (Fig. 2B). IGFBP-4 mRNA was elevated from day 2 after onset of loading and remained elevated (Fig. 2C), whereas IGFBP-5 was the only parameter to display a decrease on day 8, which then returned to control levels by day 16 (Fig. 2D).


Figure 2
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  		Fig. 2. Effect on the mRNA expression of insulin-like growth factor-I (IGF-I; A), mechano-growth factor (MGF; B), insulin-like growth factor binding protein (IGFBP)-4 (C), and IGFBP-5 (D) mRNA during 16 days of mechanical loading of the plantaris tendon in rats. All mRNA values were measured by semiquantitative RT-PCR using 18S RNA as an internal standard. For sufficient material, the control samples were pooled in groups of 4 and the loaded tendon samples were pooled in pairs. Data are means ± SD of n = 4/group at each time point. *P < 0.05 vs. control.

 
Tendon collagen system.   The precursor for the most abundant collagen protein in a normal tendon, procollagen I alpha 2 mRNA, displayed a significant rise at day 8 after onset of loading and stayed elevated at day 16 (Fig. 3A).


Figure 3
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  		Fig. 3. Effect on the mRNA expression of procollagen I alpha 2 and procollagen III alpha 1 mRNAs during 16 days of mechanical loading of the plantaris tendon in rats. All mRNA values were measured by semiquantitative RT-PCR using 18S RNA as an internal standard. A: results for procollagen I alpha 2 (ProCol1A2) mRNA. B: results for procollagen III alpha 1 (ProCol3A1) mRNA. For sufficient material, the control samples were pooled in groups of 4 and the loaded tendon samples were pooled in pairs. Data are means ± SD of n = 4/group at each time point. *P < 0.05 vs. control.

 
The mRNA for the precursor of collagen III protein, procollagen III alpha 1, was already increased by 2 days of loading and it remained significantly elevated during the remaining experimental period (Fig. 3B).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The primary goal of the present study was to describe the relationship between the IGF-I system and collagen expression in the tendon tissue during increased mechanical loading. We observed an upregulation of expression of IGF-I, IGFBP-4, and procollagen mRNA in association with an increase in tendon mass, whereas IGFBP-5 mRNA decreased. The presence of the IGF-I isoform, MGF, was demonstrated and observed to be upregulated in the loaded tendon. This adds knowledge to our understanding of tendon tissue's reaction to loading and thereby also gives an understanding of the pathogenesis of overuse injuries in tendons.

In accordance with previous findings, we found that mechanical loading of the tendon results in an enlargement of the tendon (9, 42). This could be due to edema of the tendon and thus an increased water content. The reason for edema would be that the loading of the tendon exceeds the tendon's mechanistic capacity and thereby gives raise to an inflammatory reaction. In support to this hypothesis we observed that procollagen type III mRNA is upregulated on day 2, whereas a significant increase for procollagen type I mRNA is detected on day 8. Both collagen type I and III mRNA are known to be upregulated, with a preference for the latter in overloaded and ruptured tendon (26, 44). However, a histological study using a similar loading method of the plantaris tendon in rats did not demonstrate any signs of inflammatory reaction but detected hypertrophied fibroblasts with electron microscopic changes correlated to increased secretion of collagen (46).

A translation to collagen protein of the increase in procollagen mRNA could also explain the increased tendon mass observed. To support this hypothesis, an increase in tendon collagen synthesis has recently been shown to occur in human tendon within the first days after a bout of acute exercise (32, 37) Furthermore, enhanced physical exercise in rodents has demonstrated a significant increase in the number, diameter, and cross-sectional area of collagen fibrils after 1 wk (36). However, a measurement of the collagen content or collagen synthesis is needed to state that the tendon enlargement was due to increased collagen protein.

MGF has been demonstrated in exercised and/or damaged muscle, and changes in the cytoskeleton have been proposed to be involved in the transduction mechanism. McKoy et al. (35) demonstrated a markedly increased expression of beta-actin, a cytoskeleton protein gene, together with an expression of MGF in stretched muscle. In contrast, muscle deficient of another cytoskeleton protein, dystrophin, is unable to express MGF in response to strain (19). Zamora and Marini (46) demonstrated that membrane renewal of the tendon fibrocytes is one of the observations seen in loading of the plantaris tendon and thus changes in the cytoskeleton of the fibrocytes is plausible. In muscle, MGF is believed to be important for initiating satellite cell activation and immediately increasing protein synthesis, whereas the later upregulation of IGF-IEa is responsible for a more sustained upregulation of the protein synthesis (24). We were able to demonstrate that loaded plantaris tendon tissue is capable of upregulating MGF mRNA with a faster response than IGF-I mRNA, similar to findings in loaded skeletal muscle (18, 20). There is no knowledge about MGF being capable of stimulating collagen production from fibroblasts as seen for IGF-I, but in muscle there is an anabolic effect on the protein synthesis for both MGF and IGF-IEa (18, 25). It could be speculated that MGF in the tendon is responsible for the early increase in collagen expression in response to the mechanical stimuli. However, further studies are needed. Although the exact role of MGF cannot be stated from this study, it is clear that tendon tissue can increase its expression of MGF in response to mechanical loading and that this increase occurs earlier than the IGF-I expression.

Expression of mRNA for IGF-I in tendon was not increased until 8–16 days after the onset of the loading regimen. This is an increased response that is somewhat delayed compared with the time pattern seen in plantaris muscle (20) but one that appears to parallel the upregulation of tendon procollagen type I mRNA expression. It cannot be excluded that, in the present study, we underestimate the immediate effect of loading on IGF-I mRNA expression, as we have not determined the degradation of IGF-I mRNA. In line with this perspective, Dahlgren et al. (14) found that tendon injury initially resulted in a decrease in IGF-I peptide in the tendon tissue, and they hypothesized an increased degradation of IGF-I as a reflection of the degree of tissue destruction. However, they did not see a decrease in mRNA expression of IGF-I (14). Finally, the pooling of the tendons before performing RT-PCR as well as the restricted number of samples increases the risk of a type II statistical error and thus an underestimation of small changes within the measured time points.

We found a similar time pattern for the increased expression of IGFBP-4 to that observed for procollagen type III with a rapid and sustained increase in mRNA. Interestingly, this fits with the observations that IGFBP-4 is elevated in skeletal muscle using a similar experimental model (4) and that this elevation in muscle is likely due to a stimulation of the intramuscular connective tissue, because IGFBP-4 has a predominant localization in connective tissue (10). In vivo obtained data from our laboratory support such a possibility, because we observed an increase in IGFBP-4 peptide in the connective tissue surrounding the human Achilles tendon when mechanically loaded with a 36-km run (Olesen et al., unpublished observations). Several studies have shown that IGFBP-4 principally inhibits the IGF-I effect (28, 39), but large concentrations of IGFBP-4 have been demonstrated to result in an increased bioavailability of IGF-I (38). In theory, increased IGFBP-4 protein locally in the tendon could enhance the anabolic effect of IGF-I and thereby be able to strengthen the tendon.

The precise role of IGFBP-4 in the tendon tissue was not elucidated in this experiment. However, two different hypotheses can be made. First, one can view the elevation as a local response of the tendon fibroblasts to an increased stimulation, where IGFBP-4 would decrease the anabolic effect of IGF-I on the connective tissue due to binding of IGF-I. Second, an increased level of IGFBP-4 in the loaded tendon could be responsible for a local anabolic effect of IGF-I on collagen synthesis with emphasis on collagen type III owing to the fact that an elevated level of IGFBP-4 protein has the possibility to increase the bioavailability of IGF-I.

Similar to IGFBP-4, IGFBP-5 mRNA has been shown to be expressed in connective tissue (11). In skeletal muscle IGFBP-5 mRNA expression appears to be influenced by the loading amount on the tissue in an opposite manner to IGFBP-4 with an upregulation during immobilization and a decease with loading (4, 8, 21). In accordance with the findings in skeletal muscle, we report a decrease in the tendon IGFBP-5 mRNA levels at day 8 after increased load; these levels returned back to normal by day 16. We have no definitive explanation for the time course of the observed response.

In conclusion, we have demonstrated that increased loading of tendon tissue results in an upregulation of expression of IGF-I, IGFBP-4, and procollagen mRNA and that this is associated with an increase in tendon mass. Furthermore, the presence of the IGF-I isoform MGF was demonstrated in tendon, and MGF displayed an early upregulation of its expression with mechanical loading. Because the findings are descriptive and at mRNA level, further studies are required to define the relationship between IGF-I and collagen production in the tendon. However, a role for the IGF-I system in collagen synthesis in tendon in response to mechanical loading stimuli is suggested.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by grants from the Danish Rheumatism Association, Clinical Institute, Aarhus University. The Danish Medical Research Council (22010154) and the National Space Biomedical Research Institute (NCC9-58-70).


   ACKNOWLEDGMENTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors address special thanks to Sam McCue, Anqi Qin, Li Zhang, and Daniel C. Cheng for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Olesen, Institute of Sports Medicine, Bispebjerg Hospital, DK-2400 Copenhagen, Denmark (e-mail: olesenjens{at}yahoo.dk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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