DNAJB1-PRKACA fusion drives fibrolamellar liver cancer through impaired SIK signaling and CRTC2/p300-mediated transcriptional reprogramming.

Fibrolamellar carcinoma (FLC) is a liver cancer of adolescents and young adults characterized by fusions of the genes encoding the protein kinase A catalytic subunit, PRKACA, and heat shock protein, DNAJB1. The chimeric DNAJB1-PRKACA protein has increased kinase activity and is essential for FLC xenograft growth. Here, we explore the critical oncogenic pathways controlled by DNAJB1-PRKACA using patient-derived FLC models, engineered systems, and patient samples. We show that a core function of DNAJB1-PRKACA is the phosphorylation and inactivation of Salt-inducible kinases (SIKs). This leads to deregulation of the CRTC2 transcriptional co-activator and p300 acetyltransferase, resulting in transcriptional reprogramming and increased global histone acetylation, driving malignant growth. Our studies establish a central oncogenic mechanism of DNAJB1-PRKACA and suggest the potential of targeting CRTC2/p300 in FLC. Notably, these findings link this rare cancer's signature fusion oncoprotein to more common cancer gene alterations involving STK11 and GNAS, which also function via SIK suppression.

Partial prevention of glucocorticoid-induced osteocyte deterioration in young male mice with osteocrin gene therapy.

Glucocorticoid excess suppresses osteocyte remodeling of surrounding bone minerals, causes apoptosis of osteoblasts and osteocytes, and disrupts bone remodeling, eventually, leading to glucocorticoid-induced osteoporosis and bone fragility. Preventing apoptosis and preserving osteocyte morphology could be an effective means of preventing bone loss during glucocorticoid treatment. We hypothesized that osteocrin, which preserves osteocyte viability and morphology in Sp7-deficient mice, could prevent osteocyte death and dysfunction in a glucocorticoid excess model. We used adeno-associated virus (AAV8) to induce osteocrin overexpression in mice one week before implantation with prednisolone or placebo pellets. After 28 days, prednisolone caused the expected reduction in cortical bone thickness and osteocyte canalicular length in control AAV8-treated mice, and these effects were blunted in mice receiving AAV8-osteocrin. Glucocorticoid-induced changes in cortical porosity, trabecular bone mass, and gene expression were not prevented by osteocrin. These findings support a modest therapeutic potential for AAV8-osteocrin in preserving osteocyte morphology during disease.

Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Dnmt3a-mutated clonal hematopoiesis promotes osteoporosis.

Osteoporosis is caused by an imbalance of osteoclasts and osteoblasts, occurring in close proximity to hematopoietic cells in the bone marrow. Recurrent somatic mutations that lead to an expanded population of mutant blood cells is termed clonal hematopoiesis of indeterminate potential (CHIP). Analyzing exome sequencing data from the UK Biobank, we found CHIP to be associated with increased incident osteoporosis diagnoses and decreased bone mineral density. In murine models, hematopoietic-specific mutations in Dnmt3a, the most commonly mutated gene in CHIP, decreased bone mass via increased osteoclastogenesis. Dnmt3a-/- demethylation opened chromatin and altered activity of inflammatory transcription factors. Bone loss was driven by proinflammatory cytokines, including Irf3-NF-κB-mediated IL-20 expression from Dnmt3a mutant macrophages. Increased osteoclastogenesis due to the Dnmt3a mutations was ameliorated by alendronate or IL-20 neutralization. These results demonstrate a novel source of osteoporosis-inducing inflammation.

PTHrP targets salt-inducible kinases, HDAC4 and HDAC5, to repress chondrocyte hypertrophy in the growth plate.

Hypertrophy of chondrocytes is a crucial step in the endochondral bone formation process that drives bone lengthening and the transition to endochondral bone formation. Both Parathyroid hormone-related protein (PTHrP) and Histone deacetylase 4 (HDAC4) inhibit chondrocyte hypertrophy. Use of multiple mouse genetics models reveals how PTHrP and HDAC4 participate in a pathway that regulates chondrocyte hypertrophy. PTHrP/cAMP/protein kinase A (PKA) signaling pathway phosphorylates the PKA-target sites on salt-inducible kinase 3 (Sik3), which leads to inhibition of Sik3 kinase activity. Inhibition of Sik3 kinase activity decreases phosphorylation of HDAC4 by Sik3 at binding sites for 14-3-3; lower levels of HDAC4 phosphorylation then allow HDAC4 nuclear translocation. In the nucleus, the transcription factor, Myocyte Enhancer Factor 2 (Mef2), activates Runt-related transcription factor 2 (Runx2), and together these two transcription factors drive the hypertrophic process. HDAC4 binds both Mef2 and Runx2 and blocks their activities. There are genetic redundancies in this pathway. Sik1 and Sik2 also mediate PTHrP/cAMP/PKA signaling when Sik3 activity is low. HDAC5 also mediates PTHrP signaling when HDAC4 expression is low. Thus, PTHrP triggers a kinase cascade that leads to inhibition of the key transcription factors (Mef2 and Runx2) that promote chondrocyte hypertrophy.

A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.

Large G protein α-subunit XLαs limits clathrin-mediated endocytosis and regulates tissue iron levels in vivo.

Alterations in the activity/levels of the extralarge G protein α-subunit (XLαs) are implicated in various human disorders, such as perinatal growth retardation. Encoded by GNAS, XLαs is partly identical to the α-subunit of the stimulatory G protein (Gsα), but the cellular actions of XLαs remain poorly defined. Following an initial proteomic screen, we identified sorting nexin-9 (SNX9) and dynamins, key components of clathrin-mediated endocytosis, as binding partners of XLαs. Overexpression of XLαs in HEK293 cells inhibited internalization of transferrin, a process that depends on clathrin-mediated endocytosis, while its ablation by CRISPR/Cas9 in an osteocyte-like cell line (Ocy454) enhanced it. Similarly, primary cardiomyocytes derived from XLαs knockout (XLKO) pups showed enhanced transferrin internalization. Early postnatal XLKO mice showed a significantly higher degree of cardiac iron uptake than wild-type littermates following iron dextran injection. In XLKO neonates, iron and ferritin levels were elevated in heart and skeletal muscle, where XLαs is normally expressed abundantly. XLKO heart and skeletal muscle, as well as XLKO Ocy454 cells, showed elevated SNX9 protein levels, and siRNA-mediated knockdown of SNX9 in XLKO Ocy454 cells prevented enhanced transferrin internalization. In transfected cells, XLαs also inhibited internalization of the parathyroid hormone and type 2 vasopressin receptors. Internalization of transferrin and these G protein-coupled receptors was also inhibited in cells expressing an XLαs mutant missing the Gα portion, but not Gsα or an N-terminally truncated XLαs mutant unable to interact with SNX9 or dynamin. Thus, XLαs restricts clathrin-mediated endocytosis and plays a critical role in iron/transferrin uptake in vivo.

SIKs control osteocyte responses to parathyroid hormone.

Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.

Regulation of Sclerostin Expression in Multiple Myeloma by Dkk-1: A Potential Therapeutic Strategy for Myeloma Bone Disease.

Sclerostin is a potent inhibitor of osteoblastogenesis. Interestingly, newly diagnosed multiple myeloma (MM) patients have high levels of circulating sclerostin that correlate with disease stage and fractures. However, the source and impact of sclerostin in MM remains to be defined. Our goal was to determine the role of sclerostin in the biology of MM and its bone microenvironment as well as investigate the effect of targeting sclerostin with a neutralizing antibody (scl-Ab) in MM bone disease. Here we confirm increased sclerostin levels in MM compared with precursor disease states like monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM. Furthermore, we found that a humanized MM xenograft mouse model bearing human MM cells (NOD-SCID.CB17 male mice injected intravenously with 2.5 million of MM1.S-Luc-GFP cells) demonstrated significantly higher concentrations of mouse-derived sclerostin, suggesting a microenvironmental source of sclerostin. Associated with the increased sclerostin levels, activated ß-catenin expression levels were lower than normal in MM mouse bone marrow. Importantly, a high-affinity grade scl-Ab reversed osteolytic bone disease in this animal model. Because scl-Ab did not demonstrate significant in vitro anti-MM activity, we combined it with the proteasome inhibitor carfilzomib. Our data demonstrated that this combination therapy significantly inhibited tumor burden and improved bone disease in our in vivo MM mouse model. In agreement with our in vivo data, sclerostin expression was noted in marrow stromal cells and osteoblasts of MM patient bone marrow samples. Moreover, MM cells stimulated sclerostin expression in immature osteoblasts while inhibiting osteoblast differentiation in vitro. This was in part regulated by Dkk-1 secreted by MM cells and is a potential mechanism contributing to the osteoblast dysfunction noted in MM. Our data confirm the role of sclerostin as a potential therapeutic target in MM bone disease and provides the rationale for studying scl-Ab combined with proteasome inhibitors in MM. © 2016 American Society for Bone and Mineral Research.

The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro.

Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone.

MicroRNA-140 Provides Robustness to the Regulation of Hypertrophic Chondrocyte Differentiation by the PTHrP-HDAC4 Pathway.

Growth plate chondrocytes go through multiple differentiation steps and eventually become hypertrophic chondrocytes. The parathyroid hormone (PTH)-related peptide (PTHrP) signaling pathway plays a central role in regulation of hypertrophic differentiation, at least in part, through enhancing activity of histone deacetylase 4 (HDAC4), a negative regulator of MEF2 transcription factors that drive hypertrophy. We have previously shown that loss of the chondrocyte-specific microRNA (miRNA), miR-140, alters chondrocyte differentiation including mild acceleration of hypertrophic differentiation. Here, we provide evidence that miR-140 interacts with the PTHrP-HDAC4 pathway to control chondrocyte differentiation. Heterozygosity of PTHrP or HDAC4 substantially impaired animal growth in miR-140 deficiency, whereas these mutations had no effect in the presence of miR-140. miR-140-deficient chondrocytes showed increased MEF2C expression with normal levels of total and phosphorylated HDAC4, indicating that the miR-140 pathway merges with the PTHrP-HDAC4 pathway at the level of MEF2C. miR-140 negatively regulated p38 mitogen-activated protein kinase (MAPK) signaling, and inhibition of p38 MAPK signaling reduced MEF2C expression. These results demonstrate that miR-140 ensures the robustness of the PTHrP/HDAC4 regulatory system by suppressing MEF2C-inducing stimuli. © 2014 American Society for Bone and Mineral Research © 2015 American Society for Bone and Mineral Research.

Schnurri-3 regulates ERK downstream of WNT signaling in osteoblasts.

Mice deficient in Schnurri-3 (SHN3; also known as HIVEP3) display increased bone formation, but harnessing this observation for therapeutic benefit requires an improved understanding of how SHN3 functions in osteoblasts. Here we identified SHN3 as a dampener of ERK activity that functions in part downstream of WNT signaling in osteoblasts. A D-domain motif within SHN3 mediated the interaction with and inhibition of ERK activity and osteoblast differentiation, and knockin of a mutation in Shn3 that abolishes this interaction resulted in aberrant ERK activation and consequent osteoblast hyperactivity in vivo. Additionally, in vivo genetic interaction studies demonstrated that crossing to Lrp5(-/-) mice partially rescued the osteosclerotic phenotype of Shn3(-/-) mice; mechanistically, this corresponded to the ability of SHN3 to inhibit ERK-mediated suppression of GSK3ß. Inducible knockdown of Shn3 in adult mice resulted in a high-bone mass phenotype, providing evidence that transient blockade of these pathways in adults holds promise as a therapy for osteoporosis.

Control of bone resorption in mice by Schnurri-3.

Mice lacking the large zinc finger protein Schnurri-3 (Shn3) display increased bone mass, in part, attributable to augmented osteoblastic bone formation. Here, we show that in addition to regulating bone formation, Shn3 indirectly controls bone resorption by osteoclasts in vivo. Although Shn3 plays no cell-intrinsic role in osteoclasts, Shn3-deficient animals show decreased serum markers of bone turnover. Mesenchymal cells lacking Shn3 are defective in promoting osteoclastogenesis in response to selective stimuli, likely attributable to reduced expression of the key osteoclastogenic factor receptor activator of nuclear factor-κB ligand. The bone phenotype of Shn3-deficient mice becomes more pronounced with age, and mice lacking Shn3 are completely resistant to disuse osteopenia, a process that requires functional osteoclasts. Finally, selective deletion of Shn3 in the mesenchymal lineage recapitulates the high bone mass phenotype of global Shn3 KO mice, including reduced osteoclastic bone catabolism in vivo, indicating that Shn3 expression in mesenchymal cells directly controls osteoblastic bone formation and indirectly regulates osteoclastic bone resorption.

Pharmacologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice.

Drug targeting of adult stem cells has been proposed as a strategy for regenerative medicine, but very few drugs are known to target stem cell populations in vivo. Mesenchymal stem/progenitor cells (MSCs) are a multipotent population of cells that can differentiate into muscle, bone, fat, and other cell types in context-specific manners. Bortezomib (Bzb) is a clinically available proteasome inhibitor used in the treatment of multiple myeloma. Here, we show that Bzb induces MSCs to preferentially undergo osteoblastic differentiation, in part by modulation of the bone-specifying transcription factor runt-related transcription factor 2 (Runx-2) in mice. Mice implanted with MSCs showed increased ectopic ossicle and bone formation when recipients received low doses of Bzb. Furthermore, this treatment increased bone formation and rescued bone loss in a mouse model of osteoporosis. Thus, we show that a tissue-resident adult stem cell population in vivo can be pharmacologically modified to promote a regenerative function in adult animals.

Control of postnatal bone mass by the zinc finger adapter protein Schnurri-3.

The completed skeleton undergoes continuous remodeling for the duration of adult life. Rates of bone formation by osteoblasts and bone resorption by osteoclasts determine adult bone mass. Abnormalities in either the osteoblast or osteoclast compartment affect bone mass and result in skeletal disorders, the most common of which is osteoporosis, a state of low bone mass. Much is known about the molecular control of bone formation and resorption from rare single gene disorders resulting in elevated or reduced bone mass. Such genetic disorders can be attributed either to osteoclast deficiencies, collectively termed "osteopetrosis," or to intrinsically elevated osteoblast activity, termed "osteosclerosis." However, an increasing need for anabolic therapies to prevent age-induced bone loss has stimulated a search for additional genes that act at the level of the osteoblast to regulate matrix synthesis. Recently, we have discovered a zinc finger adaptor protein called Schnurri-3 (Shn3) that potently regulates adult bone mass. Mice that lack Shn3 have normal skeletal morphogenesis but display profoundly elevated bone mass that increases with age. The molecular mechanism was revealed to be the recruitment of WWP1, a Nedd4 family E3 ubiquitin ligase, by Shn3 to the major transcriptional regulator of the osteoblast, Runx2. In the absence of Shn3, Runx2 degradation by WWP1 is inhibited resulting in increased levels of Runx2 protein and enhanced expression of Runx2 target genes leading to increased osteoblast synthetic activity. Small molecules that inhibit Shn3 or WWP1 may be attractive candidates for the treatment of diseases of low bone mass.

Schnurri-3 is an essential regulator of osteoblast function and adult bone mass.

Skeletal remodelling is a cyclical process where under normal physiological conditions, bone formation occurs at sites where bone resorption has previously taken place. Homeostatic remodelling of the skeleton is mediated by osteoclasts, giant multinucleated cells of haematopoietic origin that are responsible for bone resorption and osteoblasts, which originate from mesenchymal stem cells, and synthesise the matrix constituents on bone-forming surfaces.1 Proliferation, differentiation and bone remodelling activities of these cells involve a complex temporal network of growth factors, signalling proteins and transcription factors. Dysregulation of any one component may disrupt the remodelling process and contribute to the pathogenesis of common skeletal disorders, like osteoporosis and Paget's disease. Rare single gene disorders resulting in elevated bone mass due to osteoclast defects are collectively termed osteopetrosis. Rarer still are single gene disorders, collectively termed osteosclerosis, in which elevated bone mass is due to intrinsically elevated osteoblast activity.2 While we have learned much about the molecular control of skeletal formation and remodelling from these mutations, additional genes that regulate bone mass have yet to be characterised.

Schnurri-3: a key regulator of postnatal skeletal remodeling.

Schnurri-3, a large zinc finger protein distantly related to Drosophila Shn, is a potent and essential regulator of adult bone formation. Mice lacking Shn3 display an osteosclerotic phenotype with profoundly increased bone mass due to augmented osteoblast activity. Shn3 controls protein levels of Runx2, the principal regulator of osteoblast differentiation, by promoting its degradation. In osteoblasts, Shn3 functions as a component of a trimeric complex between Runx2 and the E3 ubiquitin ligase WWP1. This complex inhibits Runx2 function and expression of genes involved in extracellular matrix mineralization due to the ability of WWP1 to promote Runx2 polyubiquitination and proteasome-dependent degradation. Our study reveals an essential role for Shn3 as a regulator of postnatal bone mass. Compounds designed to block Shn3/WWP1 function may be possible therapeutic agents for the treatment of osteoporosis.

Regulation of adult bone mass by the zinc finger adapter protein Schnurri-3.

Genetic mutations that disrupt osteoblast function can result in skeletal dysmorphogenesis or, more rarely, in increased postnatal bone formation. Here we show that Schnurri-3 (Shn3), a mammalian homolog of the Drosophila zinc finger adapter protein Shn, is an essential regulator of adult bone formation. Mice lacking Shn3 display adult-onset osteosclerosis with increased bone mass due to augmented osteoblast activity. Shn3 was found to control protein levels of Runx2, the principal transcriptional regulator of osteoblast differentiation, by promoting its degradation through recruitment of the E3 ubiquitin ligase WWP1 to Runx2. By this means, Runx2-mediated extracellular matrix mineralization was antagonized, revealing an essential role for Shn3 as a central regulator of postnatal bone mass.

Turning down the system: counter-regulatory mechanisms in bone and adaptive immunity.

Major advances have been made in recent years toward the identification of transcription factors that control cell-type-specific gene expression in the skeletal and adaptive immune systems. However, the identification of factors necessary and sufficient to drive production of effector cell proteins such as matrix components and cytokines represents the first step toward understanding how cells in bone and the adaptive system achieve their highly specialized functions. Here, we provide selected examples of counter-regulatory mechanisms that serve to turn down cells involved in extracellular matrix biosynthesis and adaptive immunity at the level of the transcription factors Runx2 and nuclear factor for the activation of T cells.

Schnurri-3 (KRC) interacts with c-Jun to regulate the IL-2 gene in T cells.

The activator protein 1 (AP-1) transcription factor is a key participant in the control of T cell proliferation, cytokine production, and effector function. In the immune system, AP-1 activity is highest in T cells, suggesting that a subset of T cell-specific coactivator proteins exist to selectively potentiate AP-1 function. Here, we describe that the expression of Schnurri-3, also known as kappa recognition component (KRC), is induced upon T cell receptor signaling in T cells and functions to regulate the expression of the interleukin 2 (IL-2) gene. Overexpression of KRC in transformed and primary T cells leads to increased IL-2 production, whereas dominant-negative KRC, or loss of KRC protein in KRC-null mice, results in diminished IL-2 production. KRC physically associates with the c-Jun transcription factor and serves as a coactivator to augment AP-1-dependent IL-2 gene transcription.