Original Full Length ArticleA distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury
Graphical abstract
Introduction
Enhancing bone formation is an important objective in medicine. In the Western world, approximately 300–400 individuals per 100,000 suffer long bone fractures each year leading to billions of dollars in health care costs [1], [2], [3]. While the majority of injuries heal within several months, approximately 5–10% of bone fractures exhibit delayed healing or a failure of the repair process [4], [5]. Although, recombinant growth factors such as bone morphogenetic proteins (BMPs) are widely used clinically in orthopedic applications to stimulate bone fusion in cases ranging from trauma to spinal fusion, recent studies suggest that supra-physiological doses are sometimes associated with adverse side effects [6], [7]. Therefore, a better understanding of regulatory mechanisms underlying the normal bone fracture response may suggest new therapeutic routes to treat skeletal injuries through increased expression of endogenous growth factors [8].
The process of skeletal repair mimics many aspects of skeletal formation [9], [10]. Mesenchymal cells initially proliferate and condense, followed by differentiation into cartilage, vascularization, and endochondral ossification of tissue at either fracture sites or in skeletal precursors. Shared patterns of gene expression further support the similarities between embryonic bone formation and adult regeneration. Transcriptional analysis of bone repair has shown that many genes are reactivated upon injury in the same temporal and spatial patterns that can be observed during earlier skeletal formation [10], [11], [12]. For example, transforming growth factor-β (TGF-β) proteins, early signals of chondrogenic condensations, appear during the inflammatory phase, followed by other markers of cartilage formation including Runx2, Wnt2, and Sox9. Although, these similar molecular and histological signatures suggest that fracture healing occurs by reactivation of a program of embryonic bone formation [11], [13], [14], the regulatory relationship between these two processes, including the mechanism that reactivates developmental signaling molecules in injured adults, is still unclear.
BMPs play important roles during the embryonic development of many tissues, including the skeleton [15]. Members of the TGF-β superfamily of secreted signaling molecules [16], BMPs are expressed within the mesenchymal stem cell anlagen that prefigure future skeletal structures [17], continue to be expressed in the perichondrium and periosteum layers surrounding growing bones [18], [19], and are upregulated following bone injury [20], [21], [22], [23]. Several BMP mutants display characteristic skeletal and soft tissue defects [24], [25], [26], providing strong genetic evidence that BMPs are endogenous signals used to control cartilage and bone formation during embryogenesis [18].
The Bmp5 locus provides particularly useful genetic tools for studying both the embryonic and adult functions of BMPs. Encoded by the classical mouse short ear (se) locus [24], Bmp5 is expressed in early mesenchymal condensations, as well as in the perichondrium and periosteum layers surrounding growing cartilage and bones [19], [27]. Bmp5 null mutants are fully viable and fertile and exhibit defects in several skeletal structures, including the pronounced shortening of the external ear pinna due to defects in the formation and growth of ear cartilages [18], [28], [29]. Importantly, Bmp5 null mutations also cause bone regeneration defects in adult animals. Following rib cage injury, the formation and maturation of cartilaginous fracture calluses are delayed in Bmp5 mutants [30]. In addition, the fracture calluses are approximately half the volume of calluses in comparably staged controls [30]. Gene expression profiles of fracture repair have shown that Bmp5 expression begins during the chondrogenic phase of bone healing and remains elevated throughout the regeneration process [12], [22]. Thus, Bmp5 is both expressed during, and required for, normal injury responses. Notably, exogenous BMP5 has been shown to have potent osteogenic capacity in vitro and in an in vivo model of fracture repair [31], [32]. Finally, recent genetic studies show that epidermal stem cell numbers and ear regeneration differences in adult animals are also linked to the Bmp5 gene [33], [34], [35], [36], suggesting that Bmp5 may play an important role in the maintenance and repair of multiple tissues.
Molecular studies of radiation and chemical mutagen-induced DNA lesions revealed several interesting alleles that disrupt Bmp5 regulation without altering the Bmp5 coding sequence [37]. Notably, two of these regulatory alleles involve structural rearrangements that occur approximately 6 kb and 105 kb downstream of the last Bmp5 exon (Fig. 1A) [38]. Bmp5 expression is lost at particular anatomical locations in the corresponding mutants, confirming that a large 3′-region is required for normal Bmp5 regulation [38]. Hundreds of kilobases (kb) of the surrounding DNA have subsequently been screened for functional enhancers using lacZ reporter genes in transgenic mice. These studies have identified multiple modular enhancers that control expression in particular developing skeletal structures, or in soft tissues [38], [39], [40]. Here we use lacZ transgenic assays to further characterize the regulatory sequences that control where and when Bmp5 is expressed during embryonic development, and during recovery from acute tissue injury in adults.
Section snippets
Plasmid and BAC clones
Mouse sequences were assembled from BAC RP23-426K2 (GenBank accession # AC079245) and BAC RP23-343K17 (GenBank accession # AC079244) [40]. Evolutionarily conserved regions (ECRs) shared between mice and humans were identified using global sequence alignment programs as previously described [41]. Genomic clones corresponding to single or small clusters of ECRs were amplified using primers listed in Supplementary Table 2. Single copy PCR products were cloned directly into the NotI site of the
Tissue specific enhancers are distributed throughout the Bmp5 genomic region
To further characterize the regulatory landscape surrounding Bmp5, global sequence alignment programs were used to compare sequences representing 418,315 bp of the mouse Bmp5 locus (GRCm38/mm10 chr9:75,797,064–76,215,378, Fig. 1A) and the corresponding human BMP5 gene locus (Materials and methods). Evolutionarily conserved regions (ECRs) greater than 100 bp showing at least 60% identity between mouse and human sequences were selected for further analysis using a lacZ transient transgenic enhancer
Discussion
Despite the importance of BMPs in development and disease, relatively little is known about the regulatory mechanisms controlling BMP activation at particular times and places during development and following injury. Here, we have characterized a set of tissue-specific enhancers that coordinate Bmp5 developmental expression. Importantly, we find that activation following adult bone or soft tissue injury occurs through a distinct regulatory region that we hereafter refer to as the ‘injury
Acknowledgments
We thank Taconic (formerly Xenogen Biosciences; NY) and the Stanford Transgenic Core for transgenic mouse production; Christie Ham for initial experiments with mouse fracture models; Namrata Barbhaiya, Vania Rashidi, and Ernst Fattakhov for help with sectioning; Ruth Tevlin for technical assistance with TRAP staining; and members of the Yang, Nusse, and Kingsley labs for helpful discussions and comments on the manuscript. This work was supported in part by a grant from the National Institutes
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- 1
Co-first authors.
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Present addresses: The Skin Care Centre, Dermatologic Surgery — 2nd floor, 835 West 10th Avenue, Vancouver, B.C. V5Z 4E8, Canada.