Scleraxis Is Essential for Tendon Differentiation by Equine Embryonic Stem Cells and in Equine Fetal Tenocytes

Abstract

The transcription factor scleraxis is required for tendon development and is upregulated during embryonic stem cell (ESC) differentiation into tenocytes. However, its role beyond early embryonic development is not defined. We utilized a short hairpin RNA to knock down scleraxis expression in ESCs and adult and fetal tenocytes. No effect on growth or morphology was observed in two-dimensional cultures. However, scleraxis knockdown in fetal tenocytes significantly reduced COL1A1, COMP, and SOX9 gene expression. Scleraxis knockdown in adult tenocytes had no effect on the expression of these genes. Strikingly, differentiating ESCs and fetal tenocytes without scleraxis failed to reorganize a three-dimensional (3D) matrix and generate artificial tendons. This was associated with a significantly reduced survival. In contrast, there was no effect on the survival and remodeling capacity of adult tenocytes following scleraxis knockdown. Overexpression of scleraxis in fetal tenocytes rescued gene expression, cell survival in 3D, and subsequent matrix contraction. Together, these results demonstrate that scleraxis is not only essential for ESC differentiation into tenocytes but that it also has an active role in maintaining fetal tenocytes, which is then redundant in adult tenocytes.

Introduction

Tendon injuries are one of the most common orthopedic injuries in human and equine athletes accounting for 46% of all limb injuries in racehorses [1]. They also occur in nonathletes and are estimated to comprise 30% to 50% of all musculoskeletal injuries in humans [2]. The long recuperation periods required following a tendon injury have a large financial impact and outcomes are often poor due to repair occurring by scar tissue formation rather than regeneration of healthy tendon tissue. The structure and function of tendons are very similar in horses and humans and they share many of the same risk factors for tendon injuries such as age and training. Horses may therefore provide a relevant large animal model for studying both the human disease process and evaluating novel therapies [3,4].

We have previously isolated [5] and characterized [6] equine embryonic stem cells (ESCs). ESCs can turn into all of the tissues of the body and therefore have the potential to provide a cell source for regenerative therapies. However, for stem cell-based therapies to be successful, it is essential that the processes which control differentiation are well understood.

Scleraxis (SCX) has a critical role in the development of force transmitting tendons [7]. It is a basic helix-loop-helix transcription factor and has been shown to directly regulate the expression of the tendon-associated genes tenomodulin [8] and collagen type I [9]. Following a tendon injury, scleraxis is upregulated [10,11], but its function and importance in tendon repair have not been established.

Scleraxis is induced in ESCs that have been injected into the injured tendon [10]. In vivo TGF-β3 is involved in scleraxis regulation and tendon formation [12] and we have demonstrated that TGF-β3 can promote a rapid upregulation of scleraxis gene expression and subsequent tendon differentiation of equine ESCs [10,13].

Scleraxis has been shown to promote the differentiation of mesenchymal stem cells (MSCs) into tenocytes [14,15], however, it has not been determined if scleraxis is essential for this process. During development, scleraxis-positive precursors can generate both tenocytes and chondrocytes [16], which indicates that scleraxis may have different roles in different cell types.

The role of scleraxis in tenocyte function beyond early embryonic development has not been described. It is known that fetal tendon tissue is able to undergo total regeneration in the absence of any scar tissue [17]. This is due to the intrinsic properties of the fetal tendon itself, as injured fetal tendons transplanted into an adult environment continue to undergo regeneration rather than scar tissue formation [18]. The mechanisms behind this are not clear, but comparisons of the mechanisms that control function in adult and fetal tenocytes may inform future therapeutic strategies to improve the regeneration of adult tendons following an injury.

The aim of this study was to determine if scleraxis is required for tendon differentiation of equine ESCs and to determine its function in fetal and adult tenocytes.

Materials and Methods

Cell culture

With the approval of the Animal Health Trust Research Ethics committee (AHT_02_2012), fetal tenocytes were isolated postmortem from a 319-day equine (thoroughbred) fetus, which had undergone spontaneous abortion. Adult tenocytes were isolated from healthy tendon tissue of an adult (thoroughbred) horse at postmortem. Tenocytes were isolated and cultured as described previously [10]. Characterized equine ESCs [6,10,13,19] were used in this study and cultured as described previously [10].

The population doubling time (DT) of the tenocytes in two-dimensional (2D) culture was performed using the formula DT = T ln2/ln(Xe/Xb), where T = incubation time, Xb = cell number at the beginning of the incubation, and Xe = cell number at the end of the incubation. Experiments were performed in triplicate and an analysis of variance was performed to determine statistically significant differences in DT.

Scleraxis knockdown

TRC2-pLKO.1-shRNA plasmids containing a shRNA specific for human SCX (clone NM_001008271.1-95s21c1; Sigma, Poole, Dorset, United Kingdom) or containing a scrambled, nontarget shRNA sequence (SHC202; Sigma) were obtained. HEK 293T cells were transfected with 1 μg of pLKO.1 plasmid along with 750 ng psPAX2 (#12260; Addgene, Cambridge, MA) and 250 ng pMD2.G (#12259; Addgene) using the FuGENE 6 transfection reagent (Promega, Hampshire, United Kingdom) according to the manufacturer's instructions. A supernatant containing infectious lentiviral particles was used to infect target cells in multiple rounds of infection at 48-h intervals in the presence of 1 μg/mL polybrene (Sigma). Five rounds of infection were used on the ESCs, and two rounds of infection were used on the fetal and adult tenocytes. This produced a high level of infection in both cell types and noninfected cells were removed by puromycin (Sigma) selection (3 μg/mL puromycin on ESCs cultured on puromycin-resistant feeders and 4 μg/mL puromycin on tenocytes).

Scleraxis overexpression

Unmodified fetal tenocytes and those that had been modified to express shSCX (1 × 10⁶ cells) were suspended in 400 μL of culture media lacking penicillin/streptomycin with 20 μg of pCMV-6-AC-GFP human SCXA (Amsbio, Abingdon, Oxfordshire, United Kingdom). Electroporation was carried out in a 4-mm gap cuvette with one 35 ms pulse of 170 V using BTX ECM830 (BTX, San Diego, CA). Selection was carried out with 800 μg/mL of G418 (Sigma) and resistant cells were expanded to create polyclonal cell lines.

Three-dimensional cell culture

Three-dimensional (3D) cell culture in collagen gels was performed as described previously [13] in both the presence and absence of 20 ng/mL TGF-β3. 3D cultures were performed for 14 days. Contraction analyses were performed using ImageJ software (National Institutes of Health) and are displayed as a percentage of the day 0 value. Cell survival in the gels was measured by digesting the constructs in 1 mg/mL type I collagenase (Sigma) for 1–2 h at 37.5°C. The remaining cells were pelleted by centrifugation, resuspended in 1 mL TrypLE Express (Invitrogen), and briefly kept at 37.5°C to fully dissociate the cells. Cell counts were performed on a hemocytometer, and results are displayed as a percentage of the seeded cell number on day 0. A Student's t-test was used to determine statistically significant differences in cell survival. Gel contraction and cell survival were performed on three independent experimental replicates with each replicate containing two to nine constructs. Modified fetal and adult tenocytes were used at P7 to P17 in these experiments. Modified ESCs were used at P18-21 in these experiments.

Immunostaining

All immunostaining was performed in triplicate. Constructs were embedded in OCT compound (VWR, Pennsylvania) and snap-frozen in liquid nitrogen-cooled isopentane. Longitudinal sections, 11 μm thick, were cut using a cryostat. The sections were fixed in 100% acetone for 10 min and stored at −20°C until used.

Tenocytes were also cultured on gelatin-coated coverslips and then fixed in 3% paraformaldehyde for 20 min at room temperature before permeabilization for 1 h with 0.1% Triton X-100.

Primary antibody incubations were carried out overnight at 4°C before detection with the fluorescently labeled secondary antibody. Primary antibodies included rabbit anti-scleraxis 1:100 (ab58655; Abcam, Cambridgeshire, United Kingdom), rabbit anti-TCF3 1:500 (ab66373; Absam), mouse anti-collagen I 1:100 (ab90395; Abcom), and rabbit anti-COMP 1:500 (kindly provided by Professor Roger Smith, Royal Veterinary College, United Kingdom). Secondary antibodies included goat anti-mouse FITC 1:200 (ab7064; Abcam) and goat anti-rabbit TR 1:200 (ab7088; Abcam). Negative controls were carried out by omitting the primary antibodies. All sections were mounted with the VECTASHIELD hardset mounting medium containing DAPI (Vector Laboratories, Peterborough, United Kingdom).

RNA extraction, cDNA synthesis, and quantitative polymerase chain reaction

Modified fetal and adult tenocytes were used between passages 9 and 14 in these experiments. RNA was extracted using 1 mL TRI Reagent (Sigma) and treated with Ambion DNA-free (Life Technologies, Paisley, United Kingdom) according to the manufacturer's instructions. cDNA was made from 1 μg of RNA using Moloney murine leukemia virus reverse transcriptase (Promega) and oligo (dT) and random hexamers as primers (both Promega). Two microliters of aliquots of cDNA was used in quantitative polymerase chain reaction (qPCR). Primers were designed using primer3 (http://frodo.wi.mit.edu/primer3) and mfold (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form) programs to obtain amplicons with a melting temperature (Tm) of 58°C–62°C, devoid a secondary structure at Tm 60°C and with an amplicon size of 50–150 bp. Primer sequences can be found in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/scd). Primers were from Sigma. qPCR was carried out using SYBR Green containing supermix (Bioline, London, United Kingdom) on a Quantica machine (Techne, Staffordshire, United Kingdom). All PCRs were performed in duplicate. PCR cycle parameters were 95°C 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s. At the end of the program, the temperature was reduced to 65°C and then gradually increased by 1°C increments up to 95°C to produce a melt curve. Gene expression was normalized to 18s rRNA gene expression levels, which did not change between treatments (data not shown), using the 2^−ΔΔCt method [20]. A Student's t-test was used to determine statistically significant fold changes in gene expression. qPCR was performed on three replicates.

Western blot

To confirm that the TCF3/E2A (Abcam) antibody could recognize the equine protein, a western blot was performed. Whole cell extract was isolated from homogenized healthy adult equine tendon tissue (taken at postmortem from horses euthanized for reasons unrelated to this study) by three rounds of freeze–thaw in extraction buffer (20 mM Hepes pH7.9, 450 mM NaCl, 0.4 mM EDTA, 25% glycerol, 1 mM PMSF), and supernatants were collected by centrifugation. Twenty micrograms of denatured protein was run on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to a PVDF membrane. Membranes were blocked and incubated with rabbit anti-TCF3 1:3,000 (Abcam) for 1 h at room temperature, followed by washing and incubation for 1 h at room temperature with a swine anti-rabbit HRP secondary antibody (1:1,000; Dako, Ely, Cambridgeshire). Immunoreactivity was detected using the ECL plus detection system (Amersham, Buckinghamshire, United Kingdom).

Results

Scleraxis knockdown has no effect on the 2D growth of equine tenocytes and undifferentiated ESCs

Overexpression of a short hairpin RNA to scleraxis (shSCX) significantly reduced endogenous scleraxis expression to undetectable levels in both adult and fetal tenocytes cultured in 2D (Fig. 1A). There was no significant difference in scleraxis expression between fetal and adult tenocytes in 2D culture. Scleraxis knockdown was further confirmed in adult tenocytes cultured in 3D at both the mRNA and protein levels (Fig. 1B, C). In 2D culture, expression of shSCX had no effect on the proliferation or morphology of either adult or fetal tenocytes in comparison to cells that expressed a shRNA containing a scrambled, nontarget sequence or unmodified control cells (Fig. 2). Undifferentiated ESCs do not express scleraxis [10] and exhibited no differences in their proliferation, morphology, or expression of pluripotency markers following scleraxis knockdown (data not shown).

FIG. 1.

Scleraxis expression can be successfully knocked down using shRNA. (A) Adult and fetal tenocytes cultured in 2D have undetectable levels of scleraxis mRNA following viral transduction with a specific shRNA to scleraxis (shSCX). This is significantly different to cells transduced with a control scrambled shRNA (nontarget), which have detectable levels of scleraxis gene expression. ND = expression not detected. *P < 0.05 using a two-tailed Student's t-test. The mean of three independent replicates is shown. Error bars represent SEM. (B) Adult tenocytes expressing shSCX and cultured in 3D collagen gels for 7 days continue to have undetectable levels of scleraxis mRNA. The result from a single experiment is shown. ND = not detected. (C) Immunocytochemistry confirms an absence of SCX protein (red) production by adult tenocytes in 3D collagen gels when expressing shSCX, but there is no effect on cartilage oligomeric matrix protein (COMP), shown in red. Collagen type I (COL1A1) staining of the matrix is shown in green. DAPI staining of the nuclei is shown in blue. Scale bar = 40 μm. 2D, two-dimensional; 3D, three-dimensional; SEM, standard error of the mean. Color images available online at www.liebertpub.com/scd

FIG. 2.

Knockdown of Scleraxis has no effect on tenocyte proliferation and morphology in 2D culture. Population doubling time and H&E staining of (A) fetal and (B) adult tenocytes show no significant differences in control nontransduced cells, cells transduced with a scrambled, nontarget shRNA (N-T), and cells transduced with a shRNA specific to scleraxis (shSCX). The mean of four independent replicates is shown. Error bars represent the SEM. Scale bar = 40 μm. H&E, hematoxylin and eosin. Color images available online at www.liebertpub.com/scd

Scleraxis knockdown has differential effects at different stages of development when cells are cultured in 3D

We have previously demonstrated the use of a 3D culture system to drive the differentiation of ESCs into a tenocyte fate [13]. When ESCs and fetal tenocytes that express shSCX are cultured in 3D, they are unable to contract the matrix and fail to generate artificial tendons. In contrast, adult tenocytes expressing shSCX behaved as normal, contracting the collagen gel to the same degree as control cells expressing a nontarget, scrambled shRNA (Fig. 3A). The data shown are from 3D cultures in the absence of TGF-β3, but the same effect on contraction was observed when the cells were cultured in the presence of 20 ng/mL TGF-β3 (data not shown).

FIG. 3.

Knockdown of Scleraxis in fetal tenocytes and ESCs prevents the contraction of 3D collagen gels by decreasing cell survival, but Scleraxis knockdown has no effect in adult tenocytes. (A) Adult tenocytes expressing a shRNA specific to scleraxis (shSCX) can contract a collagen matrix to the same degree as control cells expressing a nontarget, scrambled shRNA (N-T), ∼13% of their starting size. Fetal tenocytes and ESCs expressing shSCX cannot contract the gels beyond 90% of their starting size over a 14-day time period. The mean of four to eight independent replicates is shown. Error bars represent the SEM. Scale bar = 15 mm. (B) The expression of a shRNA to scleraxis (shSCX) significantly reduces the survival of fetal tenocytes and ESCs at 3, 7, and 14 days of culture, compared to cells that are expressing a scrambled, nontarget shRNA (N-T). *P < 0.05 using a two-tailed Student's t-test to compare N-T to shSCX for each time point. The mean of four to eight independent replicates is shown. Error bars represent the SEM. ESCs, embryonic stem cells. Color images available online at www.liebertpub.com/scd

The failure to contract the collagen gel results from a significantly reduced survival of fetal tenocytes and ESCs expressing shSCX in comparison to the nontarget cells after 3, 7, and 14 days of culture. The survival of adult tenocytes is unaffected by shSCX expression with between 69% and 106% of cells surviving at all three time points (Fig. 3B). The data shown are from 3D cultures in the absence of TGF-β3, but the same effect on cell survival was observed when the 3D constructs were cultured in the presence of 20 ng/mL TGF-β3 (data not shown).

Expression of COL1A1, COMP, and SOX9 is significantly reduced following scleraxis knockdown in fetal tenocytes but not in adult tenocytes in 2D culture

Quantitative PCR performed on adult and fetal tenocytes expressing shSCX or a nontarget shRNA demonstrated that there is no effect on the expression of any of the tendon-associated genes studied in adult tenocytes (Fig. 4B). Although the expression of the cartilage-associated gene SOX9 appears to be reduced following shSCX expression in adult tenocytes, this difference is not statistically significant. In contrast in fetal tenocytes, the expression of COL1A1, COMP, and SOX9 is significantly reduced in response to shSCX expression. The expression of TNC is unaffected and both TNMD and THBS4 have undetectable mRNA levels in shSCX and control cultures (Fig. 4A).

FIG. 4.

Tendon gene expression in scleraxis knockdown tenocytes cultured in 2D. (A) Fetal tenocytes expressing a shRNA to scleraxis (shSCX) have significantly reduced gene expression levels of COL1A1, COMP, and SOX9, compared to control cells expressing a scrambled, nontarget shRNA (nontarget). (B) Adult tenocytes expressing shSCX have no significant changes in their gene expression compared to control nontarget cells. ND = expression not detected. *P < 0.05 using a two-tailed Student's t-test. The mean of three independent replicates is shown. Error bars represent the SEM and the relative expression is plotted on a log10 scale.

TCF3/E2A protein is present in both adult and fetal tenocytes

E12 and E47 have been reported to be binding partners of scleraxis [21,22]. They are alternative splice variants of the TCF3/E2A gene. Immunocytochemistry demonstrates that TCF3 is present in both fetal and adult tenocytes (Fig. 5A), with western blot confirming that the antibody used cross-reacts with equine TCF3 (Fig. 5B).

FIG. 5.

Adult and fetal tenocytes both express TCF3/E2A protein. (A) Immunocytochemistry confirms the presence of TCF3/E2A protein in both adult and fetal tenocytes. DAPI staining of the nuclei is shown in blue. TCF3/E2A staining is shown in red. Scale bar = 40 μm. (B) Western blot confirms that the antibody is able to detect the equine protein by detecting a single band of around 70 kDa in whole cell extract taken from adult tenocytes. Color images available online at www.liebertpub.com/scd

Overexpression of scleraxis cDNA restores the ability of fetal tenocytes to remodel a 3D collagen gel by increasing cell survival

Overexpression of human SCX in unmodified fetal tenocytes and fetal tenocytes expressing shSCX was performed by stable transfection of the cells with a plasmid encoding human SCXA.

Figure 6A shows that overexpression of SCX in unmodified fetal tenocytes had no significant effect on their behavior in 3D culture, with the cells contracting the 3D collagen gel as normal. Overexpression of SCX in shSCX fetal tenocytes rescues the knockdown phenotype and restores the ability of the SCX knockdown cells to remodel a collagen gel with no significant differences observed to control cells.

FIG. 6.

Overexpression of SCX in knockdown fetal tenocytes rescues their ability to survive and remodel a 3D collagen gel. (A) Fetal tenocytes expressing a shRNA to scleraxis (shSCX) and cultured in 3D fail to contract a collagen gel. Overexpression of human SCX in these cells (shSCX+SCX) results in contraction levels, which are not significantly different to that of fetal tenocytes either expressing a control, nontarget shRNA (N-T) or only overexpressing SCX (+SCX). The mean of five to eight independent replicates is shown. Error bars represent the SEM. (B) Fetal tenocytes expressing a shRNA to scleraxis (shRNA) and cultured in 3D have significantly lower survivals than control, nontarget shRNA expressing cells (NT). Overexpression of scleraxis (+SCX) in wild-type cells or in the knockdown cells (shSCX+SCX) returns survival levels at all time points back to that of control, nontarget cells. *P < 0.05 using a two-tailed Student's t-test. The mean of eight independent replicates is shown. Error bars represent the SEM.

The overexpression of SCX in shSCX cells significantly increases their survival to levels that are not significantly different to control nontarget cells or control cells overexpressing SCX (Fig. 6B).

Overexpression of scleraxis in fetal tenocytes increases tendon gene expression in 2D cultures and rescues tendon gene expression in shSCX expressing cells

Fetal tendon cells overexpressing human SCX have significantly higher levels of expression of SCX, TNC, COMP, and SOX9. COL1A1 expression was also increased, although due to high levels of variation in expression, this increase was not significant (Fig. 7A). TNMD and THBS4 expression remains undetectable following SCX overexpression.

FIG. 7.

SCX overexpression restores gene expression in knockdown fetal tenocytes. Fetal tenocytes overexpressing human SCX (+SCX) express significantly higher levels of SCX, TNC, COMP, and SOX9 than control cells expressing a scrambled, nontarget shRNA. With the exception of SCX, overexpression of human SCX in fetal tendon cells expressing a shRNA to SCX (shSCX+SCX) has no significant difference in their gene expression levels compared to cells only overexpressing SCX (+SCX). ND = expression not detected. *P < 0.05 using a two-tailed Student's t-test. The mean of three independent replicates is shown. Error bars represent the SEM and the relative expression is plotted on a log10 scale.

Overexpression of human SCX in fetal tendon cells, which express shSCX, rescues the expression of COL1A1, COMP, and SOX9 to levels that are not significantly different to SCX expressing alone cells. TNMD and THBS4 expression remains undetectable in both cell populations and SCX expression is significantly higher in cells overexpressing SCX in the absence of shSCX coexpression (Fig. 7B).

Discussion

Scleraxis is a bHLH transcription factor required for the generation of force transmitting tendons in mice [7]. Equine ESCs have the potential to be used clinically to help regenerate tendon tissue following an injury, but understanding the mechanisms that control their differentiation is a key step in moving them toward the clinic. In previous studies, we demonstrated that equine ESCs can differentiate into tenocytes in response to TGF-β3 and 3D culture [10,13]. Differentiation was associated with an increase in scleraxis in 2D culture [10], 3D culture [13], and following in vivo injection into the injured horse tendon [10]. In this study, our aim was to determine if scleraxis is required for tendon differentiation by equine ESCs.

We used a short-hairpin RNA against human scleraxis (shSCX) and demonstrated that this could successfully knock down equine scleraxis mRNA and protein to undetectable levels. Undifferentiated equine ESCs expressing shSCX exhibited no change in growth, morphology, or marker expression in 2D culture. This was as expected as undifferentiated ESCs do not express detectable levels of SCX [10]. Fetal and adult equine tenocytes do express scleraxis, but we found that, in both cell types, scleraxis knockdown did not affect cell morphology or growth in 2D culture.

To promote high levels of tenocyte differentiation by the ESCs, we cultured them in 3D collagen gels in the presence and absence of TGF-β3, as we have previously demonstrated that 3D culture and TGF-β3 synergistically promote tendon differentiation [13]. Culture of control ESCs in 3D collagen gels for a period of 2 weeks results in the formation of “artificial tendons,” which have contracted and display aligned cells and collagen fibers along with production of other tendon matrix-associated proteins. ESCs expressing shSCX failed to contract the collagen gel at all, which was likely due to a significant reduction in survival of the ESCs expressing shSCX in these conditions compared to ESCs expressing a control, nontarget shRNA sequence. This demonstrates that ESCs do require scleraxis for functional tendon differentiation.

Interestingly, when shSCX fetal tenocytes were used in the 3D cultures, they too exhibited a significant reduction in their survival and a total failure to contract the 3D collagen gel. However, shSCX adult tenocytes behaved as normal and contracted the collagen matrix to control levels. We were able to demonstrate that the effect on fetal tenocyte survival in 3D culture was specific to scleraxis knockdown as the overexpression of human scleraxis in the knockdown cells was able to restore cell survival in 3D culture and contraction of the collagen gel returned to normal. This study therefore validates the utility of our 3D culture system to assess tenocyte functionality. We have previously shown that equine iPSCs can differentiate into cells that express tenocyte markers but that they do not remodel a collagen gel in 3D [23]. The current work also highlights the importance of testing tenocyte behavior in 3D culture as the fetal tenocytes continued to proliferate as normal in 2D following SCX knockdown but were not able to survive in our 3D system.

The fetal tenocytes used in this study were from a 319-day-old fetus. The gestation period in horses is between 322 and 387 days. The tenocytes used were therefore from fetuses near the end of gestation. The precise role of scleraxis during fetal and adult development has not been defined. Scleraxis has been used to define developing tendons in E16.5-day mice [24] and it has been shown that the expression of scleraxis in developing tendons remains consistent at least up to day E19 (90% of the way through mouse gestation). We therefore propose that the fetal tenocytes used in this study represent developing tendons, but it would be of interest in future studies to determine if scleraxis knockdown has the same effects in equine fetal tenocytes isolated from earlier stages, immature animals, and MSCs.

As the shSCX fetal tenocytes and ESCs displayed such poor survival in the collagen gels, this precluded any further studies into their phenotype. We therefore examined tendon gene expression in scleraxis knockdown fetal and adult tenocytes grown in 2D culture. We found that there was no effect on adult tenocyte gene expression. This suggests that scleraxis may not be playing an active role in maintaining adult tenocyte phenotype and behavior. In contrast, we found that SCX knockdown in fetal tenocytes resulted in significant changes in tendon gene expression in 2D culture.

COL1A1 is a known target of SCX [9] and it has previously been shown that overexpression of SCX can increase both COL1A1 gene expression in tendon-derived stem cells (TDSCs) [25] and COL1A1 protein secretion (but not mRNA expression) in bone marrow-derived stem cells (BMSCs) [14]. In support of this, we have demonstrated that SCX knockdown produces a reduction in COL1A1 expression in fetal tenocytes. Furthermore, overexpression of SCX increases COL1A1 expression. Despite being a very large increase in expression, it was not a significant increase due to the large variation in expression levels of COL1A1. Overexpression of SCX in SCX-knockdown cells was able to rescue COL1A1 expression to levels similar to that seen in nonknockdown, SCX overexpressing cells. Together, these data suggest that COL1A1 expression is modulated by SCX in fetal but not adult tenocytes.

TNMD has previously been reported to be a downstream target of SCX [8]. However, in this study, we did not detect any TNMD expression in control fetal or adult tenocytes cultured in 2D. We have previously shown a lack of TNMD mRNA, despite detectable levels of protein in adult tenocytes [10], suggesting that the protein has a different rate of degradation to its mRNA [26]. Tenocytes isolated from stage 41 embryos, which overexpress SCX, upregulate TNMD expression [8]. However, in our study, overexpression of SCX in equine fetal tenocytes did not result in TNMD expression. This supports a previous study of Tan et al. who found that SCX overexpression did not increase TNMD in TDSCs [25].

Tan et al. also demonstrated that TNC is upregulated following SCX overexpression in TDSCs [25]. TNC has two E-box binding sites for SCX in its promoter. While we found that TNC expression was significantly increased in fetal tendon cells overexpressing SCX, its expression was not downregulated following SCX knockdown. This could suggest that normal TNC expression levels in fetal tendon are not being actively maintained by SCX but that when excess levels of SCX are present, SCX, or a downstream target of SCX, does upregulate TNC.

COMP is not a known target of SCX, but it does contain two of the E-box binding sites for SCX in its promoter region. We demonstrated that SCX knockdown in fetal tenocytes results in a decrease in COMP expression, and SCX overexpression increases COMP levels in fetal tenocytes. Further investigation of the regulation of COMP by SCX is therefore merited.

THBS4 expression was not detected in either adult or fetal cells cultured in 2D, and SCX overexpression in fetal tenocytes did not result in a detectable induction of THBS4 expression. Equine THBS4 does have one E-box binding site in its promoter region and its expression has been shown to be increased in tendon-derived stem cells following SCX overexpression [25]. We have previously demonstrated that THBS4 is not expressed in adult tenocytes [10] cultured in 2D, although it is expressed in both adult tenocytes and ESCs cultured in 3D [13]. Understanding if SCX targets change in 2D versus 3D culture is a topic for future research.

SOX9 is an important driver of cartilage cell differentiation [27]. Counterintuitively, we found that SOX9 expression was significantly reduced following knockdown of SCX in fetal tenocytes. Furthermore, SCX overexpression in fetal tenocytes resulted in a significant increase in SOX9 gene expression. In BMSCs, SCX overexpression was reported to cause a decrease in SOX9 expression [14]. However, in TDSCs, SCX overexpression caused an increase in the expression of SOX9 and other cartilage-associated genes [25]. SCX has also been shown to activate aggrecan gene expression in osteosarcoma cells [28] and can regulate collagen type II expression when in a complex with SOX9 [29]. It would therefore appear that SCX may have a more complex role in the specification of tendon and cartilage lineages than simply acting to promote tendon differentiation at the expense of cartilage differentiation. Identification of other genes, which are downstream targets of SCX, may shed more light on this area.

SCX can bind to DNA as a heterodimer with other bHLH proteins such as E12 [22] and E47 [21], which are alternative splice variants of the E2A/TCF3 gene. We therefore confirmed that E2A/TCF3 is present in both fetal and adult tenocytes. The different results following scleraxis knockdown in fetal and adult tenocytes are therefore not likely to be due to differences in the availability of binding partners.

Together with previous published data on tendon stem cells, progenitor cells, and MSCs, this study demonstrates that SCX has distinct roles in different stages of tendon differentiation. While SCX has been shown to be essential for tendon development [7], its role in tendon repair is less well defined. We [10] and others [11] have shown that SCX levels are increased following an adult tendon injury, but at present the identity of the cells that upregulate SCX is unknown and the functional relevance of this is yet to be determined.

Understanding the mechanisms of scleraxis function in different stages of development and in different cell types may have relevance for the development of cell-based therapies to improve tendon regeneration in horses and humans.

Footnotes

Acknowledgments

The authors are grateful to Professor Roger Smith, Royal Veterinary College, United Kingdom, for providing the rabbit anti-COMP antibody used in this study and to Ms. Emma Goodfellow, Animal Health Trust, United Kingdom, for providing the tendon tissue samples used. This work was funded by the Horserace Betting Levy Board (grant no. vet/prj/762).

Author Disclosure Statement

No competing financial interests exist.

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