Efforts to conserve biodiversity comprise a patchwork of international goals, national-level plans, and local interventions that, overall, are failing. We discuss the potential utility of applying ...the mitigation hierarchy, widely used during economic development activities, to all negative human impacts on biodiversity. Evaluating all biodiversity losses and gains through the mitigation hierarchy could help prioritize consideration of conservation goals and drive the empirical evaluation of conservation investments through the explicit consideration of counterfactual trends and ecosystem dynamics across scales. We explore the challenges in using this framework to achieve global conservation goals, including operationalization and monitoring and compliance, and we discuss solutions and research priorities. The mitigation hierarchy’s conceptual power and ability to clarify thinking could provide the step change needed to integrate the multiple elements of conservation goals and interventions in order to achieve successful biodiversity outcomes.
DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten‐eleven translocation (TET) enzyme family ...catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET‐mediated DNA hydroxymethylation during BMP‐induced C2C12 osteoblast differentiation using a novel cytosine‐based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339 led to increased 5mC at specific CpG‐rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7‐promoter‐reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET‐mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.
We have previously shown that parathyroid hormone (PTH) induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (NACA), leading to nuclear ...translocation of NACA and activation of target genes. Using ChIP-Seq against NACA in parallel with RNA-sequencing, we report the identification of Ubiquitin Specific Peptidase 53 (Usp53) as a target gene of PTH-activated NACA in osteoblasts. A binding site for NACA within the ChIP fragment from the Usp53 promoter was confirmed by electrophoretic mobility shift assay. Activity of the Usp53 promoter (- 2325/+ 238 bp) was regulated by the JUN-CREB complex and this activation relied on activated PKA and the presence of NACA. Usp53 knockdown in ST2 stromal cells stimulated expression of the osteoblastic markers Bglap2 (Osteocalcin) and Alpl (Alkaline phosphatase) and inhibited expression of the adipogenic markers Pparg and Cebpa. A similar effect was measured when knocking down Naca. During osteoblastogenesis, the impact of Usp53 knockdown on PTH responses varied depending on the maturation stage of the cells. In vivo implantation of Usp53-knockdown bone marrow stromal cells in immunocompromised mice showed an increase in osteoblast number and a decrease in adipocyte counts. Our data suggest that Usp53 modulates the fate of mesenchymal cells by impacting lineage selection.
Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into “stone.” In contrast, deletion of the genes encoding other ...members of the Src family kinase (SFK) fails to produce an osteopetrotic phenotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other SFKs. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn, and Fyn were the most highly expressed SFKs in WT osteoclasts, whereas Hck, Lck, Blk, and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src−/− osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src–Hck and Src–Lyn constructs in which the unique, SH3, SH2, or catalytic domains had been swapped. We found that the Src unique, SH3, and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src’s functionality is, in part, derived from an SH3–proximal proline-rich domain interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help clarify Src’s unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.
The transcriptional cofactor nascent polypeptide-associated complex and co-regulator α (NACA) regulates osteoblast maturation and activity. NACA functions, at least in part, by binding to Jun ...proto-oncogene, AP-1 transcription factor subunit (cJUN) and potentiating the transactivation of AP-1 targets such as osteocalcin (Bglap) and matrix metallopeptidase 9 (Mmp9). NACA activity is modulated by phosphorylation carried out by several kinases, but a phosphatase regulating NACA’s activity remains to be identified. Here, we used affinity purification with MS in HEK293T cells to isolate NACA complexes and identified protein phosphatase 1 catalytic subunit α (PP1A) as a NACA-associated Ser/Thr phosphatase. NACA interacted with multiple components of the PP1A holoenzyme complex: the PPP1CA catalytic subunit and the regulatory subunits PPP1R9B, PPP1R12A and PPP1R18. MS analysis revealed that NACA co-expression with PPP1CA causes dephosphorylation of NACA at Thr-89, Ser-151, and Thr-174. NACA Ser/Thr-to-alanine variants displayed increased nuclear localization, and NACA dephosphorylation was associated with specific recruitment of novel NACA interactants, such as basic transcription factor 3 (BTF3) and its homolog BTF3L4. NACA and PP1A cooperatively potentiated cJUN transcriptional activity of the AP-1–responsive MMP9-luciferase reporter, which was abolished when Thr-89, Ser-151, or Thr-174 were substituted with phosphomimetic aspartate residues. We confirmed the NACA–PP1A interaction in MC3T3-E1 osteoblastic cells and observed that NACA phosphorylation status at PP1A-sensitive sites is important for the regulation of AP-1 pathway genes and for osteogenic differentiation and matrix mineralization. These results suggest that PP1A dephosphorylates NACA at specific residues, impacting cJUN transcriptional activity and osteoblast differentiation and function.
During the development of the vertebrate skeleton, the progressive differentiation and maturation of chondrocytes from mesenchymal progenitors is precisely coordinated by multiple secreted factors ...and signaling pathways. The WNT signaling pathway has been demonstrated to play a major role in chondrogenesis. However, the identification of secreted factors that fine-tune WNT activity has remained elusive. Here, in this study, we have identified PI15 (peptidase inhibitor 15, protease Inhibitor 15, SugarCrisp), a member of the CAP (cysteine rich secretory proteins, antigen 5, and pathogenesis related 1 proteins) protein superfamily, as a novel secreted WNT antagonist dynamically upregulated during chondrocyte differentiation.
ATDC5 cells, C3H10T1/2 micromass cultures and primary chondrocyte cells were used as in vitro models of chondrogenesis. PI15 levels were stably depleted or overexpressed by viral shRNA or expression vectors. Chondrogenesis was evaluated by qPCR gene expression analysis and Alcian blue staining. Protein interactions were determined by coimmunoprecipitation assays.
shRNA-mediated knockdown of PI15 in ATDC5 cells, C3H10T1/2 cells or primary chondrocytes inhibits chondrogenesis, whereas the overexpression of PI15 strongly enhances chondrogenic potential. Mechanistically, PI15 binds to the LRP6 WNT co-receptor and blocks WNT-induced LRP6 phosphorylation, thus repressing WNT-induced transcriptional activity and alleviating the inhibitory effect of WNT signaling on chondrogenesis. Altogether, our findings suggest that PI15 acts as a key regulator of chondrogenesis and unveils a mechanism through which chondrocyte-derived molecules can modulate WNT activity as differentiation proceeds, thereby creating a positive feedback loop that further drives differentiation.
Hyp mice having an inactivating mutation of the phosphate‐regulating gene with homologies to endopeptidases on the X‐chromosome (Phex) gene have bones with increased matrix extracellular ...phosphoglycoprotein (MEPE). An acidic, serine‐ and aspartic acid–rich motif (ASARM) is located in the C terminus of MEPE and other mineralized tissue matrix proteins. We studied the effects of ASARM peptides on mineralization and how PHEX and MEPE interactions contribute to X‐linked hypophosphatemia (XLH). ASARM immunoreactivity was observed in the osteoid of wildtype bone and in the increased osteoid of Hyp mice. In wildtype bone, PHEX immunostaining was found particularly in osteoid osteocytes and their surrounding matrix. Treatment of MC3T3‐E1 osteoblasts with triphosphorylated (3 phosphoserines) ASARM peptide (pASARM) caused a dose‐dependent inhibition of mineralization. pASARM did not affect collagen deposition or osteoblast differentiation, suggesting that pASARM inhibits mineralization by direct binding to hydroxyapatite crystals. Binding of pASARM to mineralization foci in pASARM‐treated cultures and to synthetic hydroxyapatite crystals was confirmed by colloidal‐gold immunolabeling. Nonphosphorylated ASARM peptide showed little or no binding to hydroxyapatite and did not inhibit mineralization, showing the importance of ASARM phosphorylation in regulating mineralization. PHEX rescued the inhibition of osteoblast culture mineralization by pASARM, and mass spectrometry of cleaved peptides obtained after pASARM‐PHEX incubations identified pASARM as a substrate for PHEX. These results, showing that pASARM inhibits mineralization by binding to hydroxyapatite and that this inhibitor can be cleaved by PHEX, provide a mechanism explaining how loss of PHEX activity can lead to extracellular matrix accumulation of ASARM resulting in the osteomalacia of XLH.
Osteoblasts and adipocytes arise from a common mesenchymal precursor cell. The cell fate decision of a mesenchymal precursor cell is under the influence of molecular cues and signaling pathways that ...lead to the activation or repression of lineage-specific transcription factors. The molecular mechanisms determining osteoblast versus adipocyte lineage specificity in response to bone morphogenic protein (BMP) remain unclear. In this study, we describe the mechanism through which Zfp521 (ZNF521), a regulator of lineage progression in multiple immature cell populations, regulates lineage specification of mesenchymal progenitor cells during BMP-induced differentiation events. In vivo deletion or in vitro knockdown of Zfp521 in mesenchymal precursors resulted in increased expression of the adipocyte determinant factor Zfp423 (ZNF423). This was concurrent with the loss of histone H3K9 methylation and an increase in histone H3K9 acetylation at the Zfp423 promoter, which together are indicative of decreased gene repression. Indeed, we found that Zfp521 occupies and represses the promoter and intronic enhancer regions of Zfp423. Accordingly, conditional deletion of Zfp521 inhibited heterotopic bone formation in response to local injection of BMP2. In contrast, marrow adiposity within BMP2-induced bone was markedly enhanced in Zfp521-deficient mice, suggesting that precursor cells lacking Zfp521 differentiate preferentially into adipocytes instead of osteoblasts in response to BMP2. Consistent with a cell-autonomous role of Zfp521 in mesenchymal precursors, knockdown of Zfp521 in stromal cells prevented BMP2-induced osteoblast marker expression and simultaneously enhanced lipid accumulation and expression of adipocyte-related genes. Taken together, the data suggest that Zfp521 is a cell fate switch critical for BMP-induced osteoblast commitment and identify Zfp521 as the intrinsic repressor of Zfp423 and hence of adipocyte commitment during BMP-induced mesenchymal precursor differentiation.
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