YAP regulates periosteal expansion in fracture repair

Abstract

Bone fracture repair initiates by periosteal expansion. The periosteum is typically quiescent, but upon fracture, periosteal cells proliferate and contribute to bone fracture repair. The expansion of the periosteum is regulated by gene transcription; however, the molecular mechanisms behind periosteal expansion are unclear. Here, we show that Yes-Associated Protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) mediate periosteal expansion and periosteal cell proliferation. Bone fracture increases the number of YAP-expressing periosteal cells, and deletion of YAP and TAZ from Osterix (Osx) expressing cells impairs early periosteal expansion. Mechanistically, YAP regulates both 'cell-intrinsic' and 'cell-extrinsic' factors that allow for periosteal expansion. Specifically, we identified Bone Morphogenetic Protein 4 (BMP4) as a cell extrinsic factor regulated by YAP, that rescues the impairment of periosteal expansion upon YAP/TAZ deletion. Together, these data establish YAP mediated transcriptional mechanisms that induce periosteal expansion in the early stages of fracture repair and provide new putative targets for therapeutic interventions.

Figure 1:. YAP/TAZ deletion from Osx expressing cells impairs periosteal expansion

A. Schematic for evaluation of periosteal expansion at 4 Days Post Fracture (DPF). B. Timeline for mouse femoral osteotomy, followed by EdU injections at 4 DPF, 3 hours prior to euthanasia. C. Staining of nuclei (DAPI), proliferating cells (EdU) and YAP protein. Quantification of D. YAP⁺ cells per length of the periosteum, E. Percentage of YAP⁺ cells in the periosteum, F. Average periosteal thickness, G. Number of cells per length of the periosteum after Osx-conditional YAP/TAZ deletion. H. Staining of nuclei (DAPI), proliferating cells (EdU) and Osterix (Osx) protein. Quantification of I. Percentage of EdU⁺ cells, J. Percentage of proliferative YAP⁺ cells, K. Osx⁺ cells per length of the periosteum and L. Percentage of Osx⁺ cells in the periosteum after Osx− conditional YAP/TAZ deletion. All scale bars are 100 μM. C. Cortical Bone, P = Periosteum, M = Muscle. Gray bars indicate levels in uninjured bones in WT mice. ‘*’: p<0.05, ‘**’: p<0.01.

Figure 2:. Establishment of a gain-of-function model to identify early YAP target gene

A. Schematic for femoral osteotomies and periosteal cell isolation followed by doxycycline treatment in vitro. Quantification of mRNA for YAP (B), Cyr61 (C) and Ctgf (D). Western blot for quantification of YAP, TAZ and Actin protein levels. Molecular weights are in kDa. F. Immunofluorescence staining for DAPI, YAP and EdU in untreated cells and YAP^CA expressing cells. G. Quantification of the fraction of EdU⁺ cells after 24 hours of YAP^CA expression. H. Schematic for periosteal cell isolation and RNA/protein isolation after 4 and 8 hours of YAP^CA expression. Quantification of mRNA of YAP (I), Cyr61 (J), CTGF (K). N. Western blot quantification of YAP/TAZ and Actin protein levels. Mol wt are in kDa. L. Immunofluorescence staining for DAPI, EdU and YAP 8 hours after YAP^CA expression. M. Quantification of the fraction of EdU⁺ cells after 8 hours of YAP^CA expression. Scale bars = 10 μM. ‘*’:p<0.05, ‘***’: p<0.001, ‘****’:p<0.0001.

Figure 3:. YAP regulates cell intrinsic and extrinsic pathways that drive periosteal expansion

A. Volcano plot indicating important genes up- or down-regulated after 8 hours of YAP^CA expression. Blue dots indicate genes with −0.3>Log2FoldChange>0.3 and p value less than 0.05. B. Heat map indicating enrichment scores for all pathways dysregulated by YAP^CA. C. 261 selected gene sets that could play a role in periosteal expansion. Individual enrichment scores of D. cell intrinsic pathways and E. cell extrinsic pathways. ‘*’ padj<0.05.

Figure 4:. YAP/TAZ deletion from Osx-expressing cells alters both cell intrinsic and extrinsic pathways.

A. Schematic demonstrating spatial distribution of Osx⁺ and Osx⁻ layers in the periosteum. B. Quantification of fluorescence intensity of Osx and Cre:GFP in WT^fl/fl and YAP/TAZcKO^Osx mice. C. Staining for Osx protein, EdU and DAPI. Dotted lines indicate the periosteum and the separation between the inner Osx⁺ layer and the outer Osx⁻ layer. Quantification of D. thickness of the Osx⁺ and Osx⁻ layers and E. Percentage of proliferative EdU⁺ cells. ‘*’:p<0.05.

Figure 5:. YAP transcribes BMP4 via TEAD in periosteal cells

A. Volcano plot of differentially accessible chromatin sites after 8 hours of YAP^CA expression. Log₂FoldChange > 2 indicates more accessible chromatin after YAP^CA. B. Results of motif analysis on differentially accessible chromatin loci. TEAD motifs were most frequently present, followed by those for Sox4/5/11, AP-1 cluster transcription factors and Smad2/3. C. Chromatin locus annotated close to Ctgf was more accessible after YAP^CA expression. D. YAP and TEAD pull down CTGF in Chromatin immunoprecipitation experiments. E. TEAD co-immunoprecipitates with YAP in fracture activated periosteal cells. F. Chromatin locus annotated close to Bmp4 was more accessible after YAP^CA expression. G. YAP and TEAD pull down Bmp4 in Chromatin immunoprecipitation experiments. H. Temporal dynamics of YAP and BMP4 expression after 1, 4, 8, 12, 24 and 48 hours of Doxycycline treatment. We observe a robust and sustained induction of Bmp4 after YAP^CA expression. I. Immunostaining for BMP4 protein in the periosteum of WT^fl/fl and YAP/TAZcKO^Osx mice. Scale bar = 100 μM. J. Schematic for periosteal cell isolation from C57BL6 mice followed by treatment with MGH-CP1 K. Treatment with MGH-CP1 results in decreased mRNA levels of Bmp4. ‘**’:p<0.01, ‘****’:p<0.0001.

Figure 6:. In vivo delivery of Bmp4 during early stages of fracture repair rescues periosteal expansion in the outer Osx⁻ layer.

A. Timeline of in vivo rmBMP4 delivery for 4 days after femoral osteotomy. B. Immunofluorescence staining of Osx with DAPI (nuclei) and EdU (proliferating cells) in the inner and outer layers of the periosteum in YAP/TAZcKO^Osx mice injected with saline (top panel) or BMP4 (bottom panel). C. BMP4 treatment increases average periosteal thickness in YAP/TAZcKO^Osx mice. D. Percentage of EdU⁺ cells remain unchanged. Gray bar indicates the range of values in injured 4DPF WT^fl/fl mice. Quantification of E. thickness of the Osx⁺ and Osx⁻ layers and F. percentage of proliferative EdU⁺ cells in each layer. Scale bar = 100 μM. ‘*’:p<0.05.

Figure 7:. BMP4 promotes collagen matrix production in the periosteum.

A. Schematic for periosteal cell isolation from C57BL6 mice at 4DPF, followed by rmBMP4 treatment in vitro for 1 or 4 days. BMP4 treatment results in a robust induction of mRNA levels of B. Id1 C. and Serpine1 both 1- and 4-days post treatment. D. Col1a1 mRNA transcripts are significantly higher after 4 days of BMP4 treatment compared to saline treated controls. E. SHG imaging of periostea in YAP/TAZcKO^Osx mice after BMP4 treatment shows increased collagen matrix deposition. F. Quantification of SHG intensity in YAP/TAZcKO^Osx mice after BMP4 treatment. Scale bar = 100 μM. ‘*’:p<0.05.