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,1
Department of
* Biomedical Engineering and
Department of Orthopaedics2 State University of New York at Stony Brook, Stony Brook, New York, USA
1Correspondence: Department of Biomedical Engineering, Psychology A Building, SUNY at Stony Brook, Stony Brook, NY 11794-2580, USA. E-mail: Michael.Hadjiargyrou{at}sunysb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: fracture • repair • healing • novel • gene
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. This hypothesis is conceptually supported by the marked similarities between embryonic development of the skeleton and its regeneration (3
4
5)
, both characterized by a multitude of cellular events (migration, adhesion, proliferation, and differentiation) that are supported by the tightly orchestrated activity of thousands of genes. Ultimately, expression patterns of these genes are regulated by specific signaling cascades induced by diverse extracellular signals (e.g., physicochemical).
To demonstrate the transcriptional complexity of bone regeneration and lend credence to our hypothesis, we performed suppressive subtractive hybridization (SSH) between RNA isolated from intact bone to that of callus from postfracture (PF) days 3, 5, 7, and 10 as a means of identifying up-regulated genes in the regenerative process (6)
. More specifically, we analyzed 3635 cDNA clones that revealed 588 known genes, 821 expressed sequence tags (ESTs), and 116 cDNAs with no known homology. The large number of known genes identified represent diverse gene families involved in cell cycle, cell adhesion and communication, cytoskeleton, extracellular matrix, growth factors/cytokines, immune/inflammation, general metabolism (enzymes), muscle, protein/processing, and degradation, RNA processing, signaling, transcriptional activation, and transport, etc., all indicating the complex, interdependent nature of the healing process. Subsequently, we generated custom cDNA microarrays and confirmed that 
90% and 80% of the subtracted known genes and ESTs, respectively, are indeed up-regulated (
2.5-fold) during the repair process (6)
.
Despite these detailed analyses, the task remains to structurally and functionally characterize the hundreds of activated genes that correspond to ESTs and presumably represent novel genes. This large number of unknown genes clearly reveals our current deficiency in the complete understanding of the molecular complexity of the repair process. It further indicates that if we wish to augment the regenerative process, especially in problematic fractures (delayed and non-unions), we need to continue our investigation into the nature of these functionally uncharacterized genes. Deciphering the structure and function of these novel genes will enable us to identify potential regulatory genes. Our laboratory recently embarked on this laborious task and we present our findings for one such novel gene, termed Mustang (musculoskeletal temporally activated novel gene).
In this report, we introduce data dealing with the molecular cloning and characterization of Mustang. Mustang encodes for a small 82 amino acid protein that localizes to the nucleus and shares high homology with similar novel proteins (as identified in EST databases) from mouse, human and cow. We find Mustang to be highly expressed during embryogenesis but, more important, inactivated in most adult tissues (with the exception of skeletal muscle and tendon). In contrast, Mustang is acutely and differentially expressed during bone regeneration. Mustang spatially localizes in mesenchymal cells of the developing limbs and tail as well as in the fracture callus, especially in periosteal osteoprogenitor cells, proliferating chondrocytes, and young active osteoblasts. Taken together, these data support the notion that this novel gene plays a role in the development and regeneration of the mammalian musculoskeletal system.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and has been used in our laboratory (1
, 2
, 6
, 8
, 9)
. A total of 28 adult male Sprague-Dawley rats (Charles River, Wilmington, MA, USA) were prepared in such a manner. One set of four animals was killed 3 days postfracture, six animals at each time point of 5, 7, and 10 days, and three animals at 14 and 21 days. After death by CO2 inhalation, the contralateral control femur from each animal as well as the fracture calluses from 22 animals were dissected free and individually processed for RNA extraction. For the remaining six animals (one from each time point of 3, 5, 7, 10, 14, and 21 days), the fractured femur was removed and processed for histochemistry.
RNA purification
Total RNA was isolated from the fracture calluses and intact bones (which included bone marrow, articular, and normal growth plate cartilage) using the ToTALLY RNA kit (Ambion, Austin, TX, USA) based on the method of Chomczynski and Sacchi (10)
. Intact femurs were initially pulverized in liquid nitrogen using a pestle and mortar, then added to the denaturing solution (guanidine thiocyanate/detergent, Ambion). In contrast, each callus was added directly to the denaturing solution. Each sample was homogenized with a polytron (Brinkmann Instruments, Westbury, NY, USA) and extracted once with phenol-chloroform-isoamyl alcohol using centrifugation (10,000 g). The aqueous phase was then transferred to a fresh tube and a 1/10 aqueous phase volume of sodium acetate solution was added. The sample was once again extracted with acid-phenol-chloroform. The aqueous phase was transferred to a fresh tube, mixed with an equal volume of isopropanol, and incubated at –20°C for at least 1 h to precipitate the RNA. Finally, the RNA was pelleted by centrifugation (10,000 g), washed with 70% ethanol, air dried, and dissolved in RNase-free water/0.1 mM EDTA. The concentration of each RNA sample was determined spectrophotometrically and the integrity of all RNA samples was monitored on agarose gels.
Reverse transcription (RT) PCR
RT-PCR was performed using the Qiagen RT-PCR kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). Primers were designed based on contig sequence and predicted open reading frame (see Fig. 2
). A 50 µL reaction was set up using the following PCR conditions: 50°C for 30 min, 94°C for 10 min, and 40 cycles of 94°C for 30 s, 65°C for 1 min, and 72°C for 1 min. The PCR products were analyzed on a 1% agarose gel. Subcloning was accomplished using the PCR-Trap Cloning kit (Gene Hunter). The cloned cDNA was then confirmed by DNA sequencing.
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Northern blot analysis
Total RNA (15–20 µg) from multiple samples was prepared, fractionated on a 1% formaldehyde/agarose gel, transferred to a nylon membrane (Nytran), and ultraviolet cross-linked according to standard procedures. In Northern analysis of RNA derived from intact bone and fracture calluses, the RNA was pooled from three different samples (n=3 per time point). cDNA probes were random primer-labeled with 32P-dCTP and hybridized to the membrane at 65°C overnight in a standard hybridization solution. After hybridization, the blot was washed in a solution of 2x SSC/1% SDS at 50°C for 30 min, 0.2x SSC/1% SDS at 50°C for 30 min, and 0.2x SSC/0.1% SDS at 65°C for 30 min. For quantitative measurement of relative expression level of Mustang, the amount of bound probe was measured by exposing the labeled filter to a PhosphorImager screen. The image was then captured using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA) and the signal intensity was measured using an Image Quant software package (version 4.0). The intensity level obtained of the Mustang probe after subtracting background was then normalized to the GAPDH signal of the same filter. The values plotted represent fold change compared with intact bone after background subtraction and GAPDH normalization. After each experiment, the membrane was stripped of probe by immersion in boiling water for 30–60 s, then used for hybridization. Thus, the same RNA membrane was used in the hybridization of both cDNA probes. Tissue blots were prepared in a similar fashion and probed with the Mustang cDNA.
Histochemistry and in situ hybridization
Preparation of tissue sections
After anesthesia, fractured femurs (PF days 3, 5, 7, 10, 14, and 21) and intact bones were carefully removed from each animal, cleared of soft tissue, fixed in 10% buffered formalin, decalcified in 5% formic acid, and embedded in paraffin (PolyFin, Polysciences, Inc., Warrington, PA, USA). Serial longitudinal sections (10 µm) were cut from each bone and either stained with safranin O-fast green for the presence of cartilage (using standard histochemical procedures) or further processed for in situ hybridization. Rat embryo sections (7 µm) were purchased from Novagen (Madison, WI, USA) and had been prepared by fixation in 4% paraformaldehyde and embedded in paraffin.
Preparation of rat Mustang RNA probe
The 246 bp coding region of rat Mustang was subcloned into PCR TRAP vector (Gene Hunter). Orientation of sense and antisense strand was determined by DNA sequence analysis, which revealed the position of the insert relative to the Sp6 and T7 promoters. The fragment was then PCR amplified using T7 and Sp6 Ribo Primers (Roche, Nutley, NJ, USA) and later purified (Roche PCR Clean-up Kit). Amplification and purification of both templates was confirmed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. In vitro transcription of each cDNA was performed using a DIG labeling kit (Roche), the cDNA template was then digested by DNase. The RNA transcripts (both riboprobes) were again washed and purified using a PCR cleanup protocol (supplied by the manufacturer). Their relative concentrations were determined by serial dot blot and DIG detection assay (Roche, DIG detection kit).
In situ hybridization
Prior to hybridization, all tissue sections (intact calluses and embryos) were thoroughly deparaffinized in xylene, washed, and rehydrated in a graded series of EtOH washes. Protein digestion was accomplished by incubation in 1N HCl, followed by incubation with varying concentrations of proteinase K (1–100 µg/mL, Roche). The sections were then acetylated with 0.5% acetic anhydride in PBS (pH 8.0) for 10 min with continuous stirring. Before hybridization, riboprobes in hybridization buffer were heated at 80°C for 3 min, followed by quick cooling in ice water. The hybridization mixture contained each riboprobe (1.0 ng/µL), 50% deionized formamide, 10% dextran sulfate, 2x SSC, 0.02% SDS, 0.01% salmon sperm DNA). The slides were incubated for 16 h at 60°C in a humid atmosphere. After hybridization, the sections were washed and the same anti-DIG detection assay was used. Finally, the sections were rinsed with tap water, mounted, viewed with a Nikon microscope, and photographed using a digital camera (Sony DC330).
Generation of enhanced green fluorescent protein fusion
To assess Mustang protein localization, a GFP-Mustang fusion protein was constructed. Primers containing unique 5' EcoRI and 3' BamHI restriction sites were designed and used to amplify the complete Mustang coding region. Agarose gel electrophoresis was used to confirm the presence of the expected 282 base pair product, which was then excised from the gel and purified (MinElute, Qiagen, Inc.). The fragment was restriction digested and ligated into the pECFP-C1 Vector (Clontech Laboratories, Inc., Palo Alto, CA, USA), which is under the control of the CMV promoter and leads to the generation of a fusion protein with Mustang fused to the carboxyl terminus of GFP. Potential GFP-Mustang plasmids were isolated from transformed cells (Plasmid Mini-Prep, Qiagen, Inc.) and screened for the presence of the Mustang cDNA insert by PCR. The correct (5'-3') orientation and frame of the Mustang insert were then verified by sequencing. Bacterial stocks containing the verified GFP-Mustang plasmid were used to isolate GFP-Mustang plasmid DNA for the subsequent transfection experiments.
Cell culture and transfection
Transient transfection studies were performed using MC3T3-E1 preosteoblastic cells (11)
maintained in log growth phase using 
-MEM supplemented with 10% FBS (Life Technologies, Grand Island, NY, USA). Cells were plated in standard 6-well tissue culture plates containing glass coverslips at an initial density of 1 x 105 cells/well and transfected with GFP-Mustang plasmid, facilitated by the addition of the transfection reagent FuGene 6 (Roche) at a 9:2 ratio (µL fugene:µg DNA). As a negative control, transfections were carried out in the same manner, using the parental pECFP-C1 vector. After 48 h, cells were washed, fixed in 4% paraformaldehyde, mounted, and imaged with phase contrast and epi-fluorescence confocal microscopy at the University Microscopy Imaging Center (University Hospital and Medical Center, SUNY Stony Brook).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To confirm this cDNA microarray data and more accurately determine changes in expression between intact bone and fracture calluses, we prepared nylon membranes with RNA isolated from intact bone (contains bone marrow, articular and normal growth plate cartilage) and PF days 3, 5, 7, 10, 14, and 21 callus, and performed Northern blot analysis using a cDNA probe derived from subcloning and sequencing the original AA943790 EST 581 bp cDNA fragment after PCR amplification. Figure 1
confirms our microarray results and clearly shows that the 1.2 kb Mustang mRNA transcript is acutely up-regulated during PF days 3–21 (Fig. 1A
). The relative levels of Mustang mRNA during the maturation of the fracture callus were accurately determined by integrated optical density measurements (normalized to those of GAPDH mRNA) and are shown in Fig. 1B
. When compared with intact bone, Mustang mRNA expression in the callus dramatically rises to a 22-fold increase by PF day 3, a staggering 54-fold by PF day 5, and 16-, 8-, 4.5-, and 4.2-fold by PF days 7, 10, 14, and 21, respectively (Fig. 1B
).
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Full-length cloning and sequence analysis
Initially we designed primers based on the original AA943790 EST sequence and subcloned the expected 581 bp cDNA fragment. After verification by DNA sequencing, we decided to clone the full-length cDNA using a bioinformatics approach, forming a "contig" (a series of overlapping homologous ESTs). Using the original AA943790 nucleotide sequence (581 bp), a contig was generated based on BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST/) searches that resulted in a 1220 bp DNA fragment (two other ESTs were used to extend the original, one at the 5' end and the other at the 3' end). Our contig criteria were very stringent, with at least a 100 bp stretch of nucleotides with >93% homology. Next, a set of specific RT-PCR primers was designed based on this contig sequence in order to experimentally obtain a smaller 1025 bp fragment containing a putative 246 bp open reading frame (ORF), as well as 5' and 3' untranslated region (UTR) sequences (includes 3' polyadenylation site). RT-PCR was subsequently performed using PF day 5 callus RNA as a template and the expected 1025 bp fragment was obtained, subcloned, and sequenced. The exact nucleotide sequence of this 1025 bp fragment confirmed the one generated by the contig approach and is shown in Fig. 2
A. This 1025 bp full-length cDNA clone contains the predicted 246 bp ORF encoding a small protein of 82 amino acids (aa) with a calculated molecular weight (MW) of 9.6 kDa (Fig. 2A
).
Once we were able to verify the ORF, we used the predicted aa sequence to search GenBank for homologous proteins. None of our BLAST searches resulted in any known homologous proteins with the exception of a mouse hypothetical protein originally isolated from skeletal muscle (accession #AJ277212). Using translated BLAST searches (tblastn) of EST databases, we were able to identify homologous EST aa sequences from other species including human, mouse, and cow. The homology between our rat Mustang aa sequence and that of these other species is shown in Fig. 2B
. The actual level of aa homology between rat and mouse, human, and bovine is 93%, 88%, and 85%, respectively. Further, the majority of aa changes detected between rat Mustang and its homologues are represented by conservative substitutions. For example, even though there are five aa substitutions between rat Mustang and its mouse homologue, four of them (S
T, SA, ED, and IV) are conservative (Fig. 2B
).
The Mustang aa sequence also revealed a classic nuclear import signal (PIKKKRPPV, aa 10-18), indicating that it is a nuclear protein (Fig. 2B
). In fact, using the PSORT II algorithm (http://psort.nibb.ac.jp), it was predicted that rat Mustang is a nuclear protein with a 94.1% reliability score. Finally, no other specific motifs were detected except for N-myristoylation, N-glycosylation, and casein kinase II phosphorylation sites. Last, the classic polyadenylation site AATAAA is also present at the 3'-end UTR (Fig. 2A
).
In vitro transcription/translation and fusion protein
To determine whether the full-length Mustang cDNA encoded for the predicted 82 aa protein described above, we used an in vitro transcription/translation assay (TNT T7 Quick Coupled Transcription/Translation System; Promega, Madison, WI, USA). Using the subcloned Mustang cDNA (from fracture callus) in correct (5'-3') and reverse (3'-5') orientation in relation to the T7 promoter, we were able to obtain the expected protein product derived from the Mustang clone only in the correct orientation (data not shown). This protein had a size of 9–10 kDa, which corresponds to the molecular mass (9.6 kDa) estimated from the predicted aa sequence.
To verify that this is indeed a nuclear protein, a gene fusion protein was created by the insertion of the Mustang cDNA into a GFP vector. Transient transfections with the GFP-Mustang construct and parental pECFP-C1 plasmids were carried out to determine the intracellular localization of Mustang within preosteoblastic MC3T3 cells. The nuclei of cells transfected with GFP-Mustang labeled brightly, with virtually no fluorescence seen in the cytoplasm, indicating active translation and nuclear import of the GFP-Mustang fusion protein (Fig. 3
A–C). In contrast, the nucleoli, sites of rRNA synthesis, as well as the nuclear envelope were devoid of any staining (Fig. 3B
, arrows and Fig. 3C
, arrowheads, respectively). Transfection with the parental vector resulted in diffuse labeling throughout the cells, demonstrating active GFP translation with no apparent subcellular localization (Fig. 3D-F
). From these results it is clear that the Mustang nuclear import signal identified through the aa analysis is present in the final protein product and directs Mustang to the nucleus.
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Tissue expression of Mustang mRNA
Since Mustang represented a novel gene, we sought to determine its distribution in adult tissues. Therefore, we extracted RNA from various organs, including intact bone, adrenal, brain, eye, heart, liver, lung, parotid, skeletal muscle, stomach, tendon, testis, thymus, thyroid, and trachea, and performed Northern blot analysis. Results from this experiment revealed robust expression only in skeletal muscle and tendon (Fig. 4
). Skeletal muscle expression was expected since that was the tissue of origin for the mouse homologue (accession #AJ277212). Lower levels of expression were detected in intact bone (consistent with results from Fig. 1
) and trachea. Other tissues (kidney, small intestine, and spleen) were screened for Mustang mRNA expression and again revealed no expression (data not shown), indicating that Mustang expression is exclusive to the musculoskeletal system.
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Spatial expression of Mustang mRNA
To reveal the cellular origin(s) of Mustang mRNA, in situ hybridization was used in conjunction with sense and antisense riboprobes and sections derived from intact bone and PF days 5 and 14 fracture callus. The PF day 5 callus was chosen because it corresponds to the highest levels of Mustang expression (see Fig. 1
) and the PF day 14 callus because it represents a period of activity for cells involved in osteogenesis, chondrogenesis, and endochondral ossification (13)
. Mustang expression in intact bone is localized in the osteogenic layer of the periosteum (Fig. 5
A). Similarly, in a PF day 5 callus, Mustang is expressed in the active periosteum but at much higher levels (Fig. 5B, C
). At higher magnification it is evident that these Mustang-expressing osteogenic cells differentiate into mature osteoblasts responsible for the formation of woven bone (Fig. 5C
, arrows). As these osteoblasts become trapped in the newly made osteoid (Fig. 5C
, red arrowheads) and further differentiate into mature osteocytes (Fig. 5C
, white arrowheads), they cease expressing Mustang. Figure 5D
shows an adjacent section hybridized with the sense control Mustang riboprobe and demonstrates no labeling, as expected.
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Next we investigated Mustang mRNA expression in a PF day 14 callus. To distinguish areas of cartilage from those of bone, sections were stained by safranin O-fast green (Fig. 6
A, C, E). Adjacent sections were hybridized with the Mustang antisense riboprobe and revealed intense labeling in proliferating chondrocytes (Fig. 6B
, arrows, Fig. 6D
, arrowheads) and active osteoblasts (Fig. 6B, F
, arrows). Again, no signal was detected in the more mature and differentiated hypertrophic chondrocytes (Fig. 6D
), osteocytes within areas of intact bone (Fig. 6B
), or newly made woven bone (Fig. 6F
).
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Embryonic mRNA expression of Mustang
Since fracture repair is essentially a recapitulation of skeletal development (with exception of inflammation and remodeling), we wanted to determine whether Mustang is expressed during embryonic bone development. Similar to our temporal and spatial expression studies with fracture calluses, rat embryos at different developmental stages (E11, E14, E16, E18, E20) were used as a source of total RNA. Northern analysis of these RNA samples revealed robust Mustang expression at all time points (data not shown). Since the RNA isolated from these samples was derived from whole embryos, we could not determine which particular tissue expresses Mustang. To address this, we performed in situ hybridization on embryonic sections derived from E16 because at this stage the limb and tail buds are being formed and elongating. Abundant expression of Mustang was detected with the antisense riboprobe in the perichondrium of the tail vertebrae (Fig. 7
C, arrowheads) and mesenchymal cells of the intervertebral discs (Fig. 7C
, arrows). Mustang expression was also detected in mesenchymal cells of developing limbs (Fig. 7D
, arrows). As with the callus, adjacent sections were stained with safranin O-fast green to reveal cartilaginous areas (for comparison with hybridized sections, Fig. 7A, B
). Last, no expression was detected with the Mustang sense riboprobe (Fig. 7E, F
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 14
, 15)
, it also serves to indicate that we have limited knowledge of how broad the transcriptional control of the repair process may be, or even identify which specific processes are the critical regulators of successful bone healing. Given the complexity of the healing process, however, there clearly are a host of genes, some known and some novel, involved in the process (6)
. Thus, identifying known molecules with unknown functions (e.g., those that have been studied in other than skeletal tissues) or novel molecules (6
, 8)
that play critical roles in the cascade of molecular events (particularly in the early stages of healing) will help us define novel strategies for the enhancement and assurance of the repair process, especially ones that later may very well define the ultimate outcome in terms of speed and integrity of bone repair.
Given the large number of EST cDNAs identified in our transcriptional profiling experiments (6)
, it became apparent that candidate selection for additional studies was crucial. Our selection process took into consideration several factors. First, SSH expression data had to be confirmed by cDNA microarray analysis, which demonstrated a >2.5-fold increase in expression. Second, the background subtracted normalized intensity value of each candidate had to be >1000 arbitrary intensity units as measured by PhosphorImager. This eliminated candidates with artificially inflated fold changes due to nonsignificant intensity values. Third, candidates were selected if the specific EST was represented by more than three clones in the subtracted library, theoretically indicating abundance and presumably (but not necessarily) a greater level of involvement in the fracture healing process. Last, the candidate EST should demonstrate early activation, especially at PF days 3 and 5. This last point was an attempt to isolate early transcriptional regulators (i.e., transcription factors, activators, repressors, etc.) or genes that may play a critical role in the initiation of processes such as inflammation, chondrogenesis, and intramembranous ossification. Several ESTs met these selection criteria, and one yielded Mustang.
In the present study, we report that Mustang is a novel gene that encodes for an 82 aa nuclear protein. This novel gene is exclusively and highly expressed during bone regeneration, especially during the early phases (PF days 3 and 5). Its expression within the fracture callus was localized in osteoprogenitor cells of the periosteum, proliferating chondrocytes, and young active osteoblasts. In addition, Mustang mRNA expression was detected during embryogenesis, consistent with the notion that fracture repair recapitulates embryonic development (5)
.
Since Mustang contains a classic nuclear import signal of PIKKKRPPV, we initially sought to verify that it is indeed a nuclear protein through use of a transcription/translation assay and, more important, transfection studies with a GFP fusion protein. Both assays generated the expected protein, and the Mustang-GFP fusion construct demonstrated nuclear localization in transient transfection of preosteoblastic MC3T3 cells. The fact that the nucleoli and nuclear envelope were devoid of staining indicates that this protein plays no role in rRNA synthesis or in the nuclear membrane; thus, it is most likely involved in activities other than "housekeeping."
When the rat full-length sequence was compared with other EST sequences, we were able to find mouse, human, and cow homologues with strong aa homology: 93%, 88%, and 85%, respectively. Extensive database searches of the completed genomic sequences of C. elegans, Zebrafish, Xenopus, Yeast, Drosophila, and Arapidopsis revealed no identifiable homologues. Further searches of EST databases containing sequences from numerous other species, including D. discoideum, chicken, rabbit, pig, several fish species, etc. (see list of species at http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html), failed to identify any homology. Both the strong conservation of sequence homology in mammals (rat, mouse, human, cow) and the inability to demonstrate the presence of Mustang in other organisms (in particular, nonmammalian vertebrates) indicate that Mustang is specific to the mammalian musculoskeletal system.
This hypothesis that Mustang is involved primarily in the process of skeletogenesis was further supported by our temporal and spatial expression analyses of embryonic development and bone regeneration (fracture repair), as described above. This data are consistent with the notion that Mustang may play a role in early differentiation of skeletal cells, as demonstrated by the presence of high Mustang mRNA levels on PF days 3 and 5. Subsequently, Mustang expression decreases dramatically as the callus matures and almost reaches the very low levels detected in intact bone by PF day 21. Again, this is consistent with the idea that multiple cell differentiation pathways are being "turned off," especially those involved in osteoblastic and chondrogenic lineages. Last, the finding that Mustang expression was not detected in any normal adult tissue except skeletal muscle and tendon suggests that Mustang is specific to the musculoskeletal system and makes the elucidation of its functional significance even more appealing.
Although the precise function of Mustang is unknown at present, its unique pattern of expression during bone development and regeneration, its absence in adult tissues (with the exception of skeletal muscle and tendon), and the fact that it is a nuclear protein all lead us to speculate that this novel gene plays an important role in early transcriptional events involved in the development and regeneration of the musculoskeletal system. More specifically, the fact that Mustang is a nuclear protein is of interest because of the strong probability it may function as a regulatory molecule. This idea stems from our knowledge that there are only a few major processes that occur in the nucleus. These processes include DNA replication, rRNA synthesis, and transcription and post-transcriptional processing. In addition, there is the nuclear envelope that plays a structural/trafficking role. If we assume that Mustang plays a role in either DNA replication, rRNA synthesis or splicing, or composition of the nuclear envelope, then we would expect it to be expressed in many different types of cells or tissues where these "housekeeping" processes take place. Our expression data argue against such an assumption since Mustang expression is not detected in most normal adult tissues and is detected only in the musculoskeletal system, early phases of the fracture callus, and during embryonic development.
Hence, we can hypothesize that Mustang plays a role in transcriptional events responsible for cell differentiation. This idea is consistent with our expression data showing that Mustang is expressed by mesenchymal cells and periosteal osteoprogenitors. Since Mustang does not possess any apparent DNA/RNA binding motifs, it may not function as a transcription or splicing factor that binds DNA or RNA per se, but could conceivably be part of a large multimeric complex of gene regulatory proteins. It is already well known that the regulatory domain(s) of transcription factors typically interact with one or several other components of a transcriptional complex, either directly or indirectly via cofactors, to mediate activation or repression of gene expression (16)
. Such complexes are known to be involved in many nuclear processes such as DNA replication, transcription, nuclear transport, and signaling.
With regard to a role in transcriptional events relevant to bone, downstream regulatory proteins (SMADs) are activated by serine/threonine kinase receptors, enter the nucleus, and function as transcriptional regulators as a result of BMP (TGF-ß superfamily) signaling. SMADs interact with one another as well as with transcription factors or coregulators and stimulate BMP or TGF-ß/activin-specific target genes (17)
. Other examples are the Groucho/transducin-like enhancer of split (Gro/TLE) and Osf2/Cbfa1, known to play a key role in osteoblast differentiation (18
19
20)
. Gro/TLE functions as a transcriptional corepressor by targeting specific gene regulatory regions through their ability to interact with different DNA binding transcription factors (21
22
23)
. Last, a more recent study showed that transcription repression activity of the basic helix loop helix factor Hes1 is regulated by the interaction of Groucho and RUNX proteins (24)
.
More work is obviously needed to verify Mustang’s true function. Nevertheless, our preliminary data indicate that Mustang represents a new gene with a probable function in transcriptional events related to cell differentiation that ultimately are involved in the development and regeneration of the mammalian musculoskeletal system.
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ACKNOWLEDGMENTS |
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Received for publication June 5, 2003. Accepted for publication September 5, 2003.
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