Skeletal abnormalities in mice with Dnmt3a missense mutations.

Overgrowth and intellectual disability disorders in humans are typified by length/height and/or head circumference ≥ 2 standard deviations above the mean as well as intellectual disability and behavioral comorbidities, including autism and anxiety. Tatton-Brown-Rahman Syndrome is one type of overgrowth and intellectual disability disorder caused by heterozygous missense mutations in the DNA methyltransferase 3A (DNMT3A) gene. Numerous DNMT3A mutations have been identified in Tatton-Brown-Rahman Syndrome patients and may be associated with varying phenotype severities of clinical presentation. Two such mutations are the R882H and P904L mutations which result in severe and mild phenotypes, respectively. Mice with paralogous mutations (Dnmt3aP900L/+ and Dnmt3aR878H/+) exhibit overgrowth in their long bones (e.g., femur, humerus), but the mechanisms responsible for their skeletal overgrowth remain unknown. The goal of this study is to characterize skeletal phenotypes in mouse models of Tatton-Brown-Rahman Syndrome and identify potential cellular mechanisms involved in the skeletal overgrowth phenotype. We report that mature mice with the Dnmt3aP900L/+ or Dnmt3aR878H/+ mutation exhibit tibial overgrowth, cortical bone thinning, and weakened bone mechanical properties. Dnmt3aR878H/+ mutants also contain larger bone marrow adipocytes while Dnmt3aP900L/+ mutants show no adipocyte phenotype compared to control animals. To understand the potential cellular mechanisms regulating these phenotypes, growth plate chondrocytes, osteoblasts, and osteoclasts were assessed in juvenile mutant mice using quantitative static histomorphometry and dynamic histomorphometry. Tibial growth plates appeared thicker in mutant juvenile mice, but no changes were observed in osteoblast activity or osteoclast number in the femoral mid-diaphysis. These studies reveal new skeletal phenotypes associated with Tatton-Brown-Rahman Syndrome in mice and provide a rationale to extend clinical assessments of patients with this condition to include bone density and quality testing. These findings may be also informative for skeletal characterization of other mouse models presenting with overgrowth and intellectual disability phenotypes.

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits.

Phenotypic heterogeneity in monogenic neurodevelopmental disorders can arise from differential severity of variants underlying disease, but how distinct alleles drive variable disease presentation is not well understood. Here, we investigate missense mutations in DNA methyltransferase 3A (DNMT3A), a DNA methyltransferase associated with overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity. We generate a Dnmt3aP900L/+ mouse mimicking a mutation with mild to moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. P900L mutants exhibit core growth and behavioral phenotypes shared across models but show subtle epigenomic changes, while R878H mutants display extensive disruptions. We identify mutation-specific dysregulated genes that may contribute to variable disease severity. Shared transcriptomic disruption identified across mutations overlaps dysregulation observed in other developmental disorder models and likely drives common phenotypes. Together, our findings define central drivers of DNMT3A disorders and illustrate how variable epigenomic disruption contributes to phenotypic heterogeneity in neurodevelopmental disease.

MicroRNA-138: an emerging regulator of skeletal development, homeostasis, and disease.

Noncoding microRNAs are powerful epigenetic regulators of cellular processes by their ability to target and suppress expression of numerous protein-coding mRNAs. This multitargeting function is a unique and complex feature of microRNAs. It is now well-described that microRNAs play important roles in regulating the development and homeostasis of many cell/tissue types, including those that make up the skeletal system. In this review, we focus on microRNA-138 (miR-138) and its effects on regulating bone and cartilage cell differentiation and function. In addition to its reported role as a tumor suppressor, miR-138 appears to function as an inhibitor of osteoblast differentiation. This review provides additional information on studies that have attempted to alter miR-138 expression in vivo as a means to dampen ectopic calcification or alter bone mass. However, a review of the published literature on miR-138 in cartilage reveals a number of contradictory and inconclusive findings with respect to regulating chondrogenesis and chondrocyte catabolism. This highlights the need for more research in understanding the role of miR-138 in cartilage biology and disease. Interestingly, a number of studies in other systems have reported miR-138-mediated effects in dampening inflammation and pain responses. Future studies will reveal if a multifunctional role of miR-138 involving suppression of ectopic bone, inflammation, and pain will be beneficial in skeletal conditions such as osteoarthritis and heterotopic ossification.

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits.

Phenotypic heterogeneity is a common feature of monogenic neurodevelopmental disorders that can arise from differential severity of missense variants underlying disease, but how distinct alleles impact molecular mechanisms to drive variable disease presentation is not well understood. Here, we investigate missense mutations in the DNA methyltransferase DNMT3A associated with variable overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity in neurodevelopmental disease. We generate a DNMT3A P900L/+ mouse model mimicking a disease mutation with mild-to-moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. We show that the P900L mutation leads to disease-relevant overgrowth, obesity, and social deficits shared across DNMT3A disorder models, while the R878H mutation causes more extensive epigenomic disruption leading to differential dysregulation of enhancers elements. We identify distinct gene sets disrupted in each mutant which may contribute to mild or severe disease, and detect shared transcriptomic disruption that likely drives common phenotypes across affected individuals. Finally, we demonstrate that core gene dysregulation detected in DNMT3A mutant mice overlaps effects in other developmental disorder models, highlighting the importance of DNMT3A-deposited methylation in neurodevelopment. Together, these findings define central drivers of DNMT3A disorders and illustrate how variable disruption of transcriptional mechanisms can drive the spectrum of phenotypes in neurodevelopmental disease.

The miR-181 family: Wide-ranging pathophysiological effects on cell fate and function.

MicroRNAs (miRNAs) are epigenetic regulators that can target and inhibit translation of multiple mRNAs within a given cell type. As such, a number of different pathways and networks may be modulated as a result. In fact, miRNAs are known to regulate many cellular processes including differentiation, proliferation, inflammation, and metabolism. This review focuses on the miR-181 family and provides information from the published literature on the role of miR-181 homologs in regulating a range of activities in different cell types and tissues. Of note, we have not included details on miR-181 expression and function in the context of cancer since this is a broad topic area requiring independent review. Instead, we have focused on describing the function and mechanism of miR-181 family members on differentiation toward a number of cell lineages in various non-neoplastic conditions (e.g., immune/hematopoietic cells, osteoblasts, osteoclasts, chondrocytes, adipocytes). We have also provided information on how modulation of miR-181 homologs can have positive effects on disease states such as cardiac abnormalities, pulmonary arterial hypertension, thrombosis, osteoarthritis, and vascular inflammation. In this context, we have used some examples of FDA-approved drugs that modulate miR-181 expression. We conclude by discussing some common mechanisms by which miR-181 homologs appear to regulate a number of different cellular processes and how targeting specific miR-181 family members may lead to attractive therapeutic approaches to treat a number of human disease or repair conditions, including those associated with the aging process.

Modulation of MicroRNA Expression During In Vitro Chondrogenesis.

Since their discovery in 1993, microRNAs (miRNAs) are now recognized as important epigenetic regulators of many mammalian cellular processes including proliferation, apoptosis, metabolism, and differentiation. These small non-coding RNAs function by interacting with specific regions in the 3'-untranslated region of mRNAs, thereby resulting in mRNA degradation or suppression of translation. Since miRNAs have the ability to target many mRNAs within a given cell type, a number of cellular pathways and networks may be regulated as a result. To study the function of miRNAs, a number of methods can be used to modulate their activity in cells such as synthetic mimics or antagomirs for short-term assays or viral-based approaches for longer-term experiments such as cell differentiation assays. In this chapter, we provide our methodology to constitutively overexpress a desired miRNA during in vitro chondrogenesis of human cartilage progenitor cells (CPCs). Specifically, we describe how we obtain CPCs from human articular cartilage specimens, how we generate and titrate lentivirus engineered to overexpress a precursor miRNA, how we transduce CPCs with lentivirus and differentiate them toward the chondrocyte lineage, and how we extract RNA and measure expression levels of the miRNA of interest during in vitro chondrogenesis. We also provide some data from our laboratory demonstrating that we can achieve and maintain miRNA overexpression for up to 14 days in cartilage pellet cultures. We predict that these lentiviral-based approaches will also be useful to study how miRNA modulation of progenitor cells affects cell differentiation and extracellular matrix production within three-dimensional biomaterial scaffolds.

Gasdermin D deficiency attenuates arthritis induced by traumatic injury but not autoantibody-assembled immune complexes.

BACKGROUND: Gasdermin D (GSDMD) is cleaved by several proteases including by caspase-1, a component of intracellular protein complexes called inflammasomes. Caspase-1 also converts pro-interleukin-1ß (pro-IL-1ß) and pro-IL-18 into bioactive IL-1ß and IL-18, respectively. GSDMD amino-terminal fragments form plasma membrane pores, which mediate the secretion of IL-1ß and IL-18 and cause the inflammatory form of cell death pyroptosis. Here, we tested the hypothesis that GSDMD contributes to joint degeneration in the K/BxN serum transfer-induced arthritis (STIA) model in which autoantibodies against glucose-6-phosphate isomerase promote the formation of pathogenic immune complexes on the surface of myeloid cells, which highly express the inflammasomes. The unexpected outcomes with the STIA model prompted us to determine the role of GSDMD in the post-traumatic osteoarthritis (PTOA) model caused by meniscus ligamentous injury (MLI) based on the hypothesis that this pore-forming protein is activated by signals released from damaged joint tissues. METHODS: Gsdmd +/+ and Gsdmd-/- mice were injected with K/BxN mouse serum or subjected to MLI to cause STIA or PTOA, respectively. Paw and ankle swelling and DXA scanning were used to assess the outcomes in the STIA model whereas histopathology and micro-computed tomography (µCT) were utilized to monitor joints in the PTOA model. Murine and human joint tissues were also examined for GSDMD, IL-1ß, and IL-18 expression by qPCR, immunohistochemistry, or immunoblotting. RESULTS: GSDMD levels were higher in serum-inoculated paws compared to PBS-injected paws. Unexpectedly, ablation of GSDMD failed to reduce joint swelling and osteolysis, suggesting that GSDMD was dispensable for the pathogenesis of STIA. GSDMD levels were also higher in MLI compared to sham-operated joints. Importantly, ablation of GSDMD attenuated MLI-associated cartilage degradation (p = 0.0097), synovitis (p = 0.014), subchondral bone sclerosis (p = 0.0006), and subchondral bone plate thickness (p = 0.0174) based on histopathological and µCT analyses. CONCLUSION: GSDMD plays a key role in the pathogenesis of PTOA, but not STIA, suggesting that its actions in experimental arthropathy are tissue context-specific.

Ablation of Proliferating Osteoblast Lineage Cells After Fracture Leads to Atrophic Nonunion in a Mouse Model.

Nonunion is defined as the permanent failure of a fractured bone to heal, often necessitating surgical intervention. Atrophic nonunions are a subtype that are particularly difficult to treat. Animal models of atrophic nonunion are available; however, these require surgical or radiation-induced trauma to disrupt periosteal healing. These methods are invasive and not representative of many clinical nonunions where osseous regeneration has been arrested by a "failure of biology". We hypothesized that arresting osteoblast cell proliferation after fracture would lead to atrophic nonunion in mice. Using mice that express a thymidine kinase (tk) "suicide gene" driven by the 3.6Col1a1 promoter (Col1-tk), proliferating osteoblast lineage cells can be ablated upon exposure to the nucleoside analog ganciclovir (GCV). Wild-type (WT; control) and Col1-tk littermates were subjected to a full femur fracture and intramedullary fixation at 12 weeks age. We confirmed abundant tk+ cells in fracture callus of Col-tk mice dosed with water or GCV, specifically many osteoblasts, osteocytes, and chondrocytes at the cartilage-bone interface. Histologically, we observed altered callus composition in Col1-tk mice at 2 and 3 weeks postfracture, with significantly less bone and more fibrous tissue. Col1-tk mice, monitored for 12 weeks with in vivo radiographs and micro-computed tomography (µCT) scans, had delayed bone bridging and reduced callus size. After euthanasia, ex vivo µCT and histology showed failed union with residual bone fragments and fibrous tissue in Col1-tk mice. Biomechanical testing showed a failure to recover torsional strength in Col1-tk mice, in contrast to WT. Our data indicates that suppression of proliferating osteoblast-lineage cells for at least 2 weeks after fracture blunts the formation and remodeling of a mineralized callus leading to a functional nonunion. We propose this as a new murine model of atrophic nonunion. © 2021 American Society for Bone and Mineral Research (ASBMR).

Biochemical and immuno-histochemical localization of type IIA procollagen in annulus fibrosus of mature bovine intervertebral disc.

For next generation tissue-engineered constructs and regenerative medicine to succeed clinically, the basic biology and extracellular matrix composition of tissues that these repair techniques seek to restore have to be fully determined. Using the latest reagents coupled with tried and tested methodologies, we continue to uncover previously undetected structural proteins in mature intervertebral disc. In this study we show that the "embryonic" type IIA procollagen isoform (containing a cysteine-rich amino propeptide) was biochemically detectable in the annulus fibrosus of both calf and mature steer caudal intervertebral discs, but not in the nucleus pulposus where the type IIB isoform was predominantly localized. Specifically, the triple-helical type IIA procollagen isoform immunolocalized in the outer margins of the inner annulus fibrosus. Triple helical processed type II collagen exclusively localized within the inter-lamellae regions and with type IIA procollagen in the intra-lamellae regions. Mass spectrometry of the α1(II) collagen chains from the region where type IIA procollagen localized showed high 3-hydroxylation of Proline-944, a post-translational modification that is correlated with thin collagen fibrils as in the nucleus pulposus. The findings implicate small diameter fibrils of type IIA procollagen in select regions of the annulus fibrosus where it likely contributes to the organization of collagen bundles and structural properties within the type I-type II collagen transition zone.

Expression profiling of mitochondria-associated microRNAs during osteogenic differentiation of human MSCs.

Small non-coding microRNAs (miRNAs) have the ability to target and bind to many mRNAs within the cytosol resulting in reduced protein expression and modulation of a number of cellular pathways and networks. In addition to the cytosol, miRNAs have been identified in other cellular compartments and organelles, including the mitochondria. While a few mitochondria-associated miRNAs (mitomiRs) are predicted to be derived from the mitochondrial genome, the majority appear to be transcribed from nuclear DNA and somehow transported into the mitochondria. These findings raise interesting questions about why miRNAs are located in the mitochondria and if they play a role in regulating processes within these organelles. Previously published work from our laboratory showed that miR-181a/b can regulate osteogenesis, in part, by enhancing mitochondrial metabolism. In other published studies, miR-181 paralogs and many other miRNAs have been identified in mitochondrial extracts derived from common cell lines and specific primary cells and tissues. Taken together, we were motivated to identify mitomiR expression profiles during in vitro osteogenesis. Specifically, we obtained RNA from purified mitochondrial extracts of human bone marrow-derived mesenchymal stem/stromal cells (MSCs) and from whole cell extracts of MSCs at day 0 or following osteogenic induction for 3, 7 and 14 days. Utilizing Affymetrix GeneChip™ miRNA 4.0 arrays, mitomiR expression signatures were determined at each time point. Based on the Affymetrix detection above background algorithm, the total number of miRNAs detected in MSC mitochondria extracts was 527 (non-induced MSCs), 627 (day 3 induced), 372 (day 7 induced) and 498 (day 14 induced). In addition, we identified significantly differentially-expressed mitomiRs at day 7 and day 14 of osteogenic induction when compared to day 0 (fold change ≥1.5; adjusted p value <0.05). In general, the most pronounced and highly significant changes in mitomiR expression during osteogenesis were observed at the day 7 time point. Interestingly, most miRNAs found to be differentially-expressed in mitochondria extracts did not show significantly altered expression in whole cell extracts at the same time points during osteoblast differentiation. This array study provides novel information on miRNAs associated with the mitochondria in MSCs during differentiation toward the osteoblast phenotype. These findings will guide future research to identify new miRNA candidates that may function in regulating mitochondrial function and/or bone formation, homeostasis or repair.

Delineation of the 1q24.3 microdeletion syndrome provides further evidence for the potential role of non-coding RNAs in regulating the skeletal phenotype.

Microdeletions within 1q24 have been associated with growth deficiency, varying intellectual disability, and skeletal abnormalities. The candidate locus responsible for the various phenotypic features of this syndrome has previously been predicted to lie in the area of 1q24.3, but molecular evidence of the causative gene remains elusive. Here, we report two additional patients carrying the smallest reported 1q24 deletion to date. Patient 1 exhibited intrauterine growth retardation, shortening of the long bones, frontal bossing, microstomia, micrognathia, and a language acquisition delay. Her mother, Patient 2, displayed a broad forehead and nasal bridge, thick supraorbital ridges, and toe brachydactyly, along with learning disability and language acquisition delay. The microdeletion encompasses a 94 Kb region containing exon 14 and portions of the surrounding introns of the gene encoding dynamin 3 (DNM3), resulting in an in-frame loss of 38 amino acids. This microdeletion site also contains a long non-coding RNA (DNM3OS) and three microRNAs (miR-214, miR-199A2, and miR-3120). Following culture of patient-derived and control fibroblasts, molecular analyses were performed to determine expression levels of genes affected by the heterozygous deletion. Results show decreased expression of DNM3OS and miR-214-3p in patient fibroblasts cultured in an osteogenic induction medium. Overall, our data provide further evidence to support a functional role for non-coding RNAs in regulating the skeletal phenotype, and the potential of a functionally-impaired DNM3 protein causing the non-skeletal disease pathogenesis.

RNA Isolation from Articular Cartilage Tissue.

Isolation of high-quality RNA directly from tissues is desirable to obtain precise information of in vivo gene expression profiles in cells embedded within their extracellular matrix (ECM). It is well known that purification of RNA from cartilage tissues is particularly challenging due to low cell (chondrocyte) content and its dense ECM rich in large negatively charged proteoglycans that can copurify with RNA. Older methodologies to purify RNA from cartilage involved the use of concentrated denaturing solutions containing guanidinium isothiocyanate followed by ultracentrifugation in cesium trifluoroacetate. Such ultracentrifugation approaches are rarely used now since the emergence of more user-friendly mini spin column chromatography kits. For this chapter, we tested and compared three methods to isolate RNA from immature murine articular (femoral head) cartilage and found that the combination of TRIzol® reagent and spin column chromatography (Norgen Total RNA Purification Kit) was the best approach to generate higher quality RNA. Here, the average RNA Integrity Number (RIN), as determined by Bioanalyzer technology, was 7.1. We then applied this method to attempt to isolate RNA directly from human articular cartilage harvested from three osteoarthritic (OA) knee joint specimens. As expected, the concentration and quality of RNA obtained differed between samples. However, from one specimen, we were able to isolate approximately 3 µg of total RNA (including small noncoding RNAs) from 100 mg of human OA cartilage with a RIN = 7.9. Despite the patient-to-patient variabilities that are known to exist between cartilage specimens from OA joints, we have demonstrated that it is possible to obtain reasonably high levels of RNA from human OA articular cartilage at a quality suitable for downstream analyses including microarray and RNA-Seq. A detailed description of our preferred RNA purification methodology, which can be used to isolate RNA from human, bovine, or rodent cartilage tissue, is provided in this chapter.

The role of microRNAs in bone development.

Epigenetic regulation is critical for proper bone development. Evidence from a large body of published literature informs us that microRNAs (miRNAs) are important epigenetic factors that control many aspects of bone development, homeostasis, and repair processes. These small non-coding RNAs function at the post-transcriptional level to suppress expression of specific target genes. Many target genes may be affected by one miRNA resulting in alteration in cellular pathways and networks. Therefore, changes in levels or activity of a specific miRNA (e.g. via genetic mutations, disease scenarios, or by over-expression or inhibition strategies in vitro or in vivo) can lead to substantial changes in cell processes including proliferation, metabolism, apoptosis and differentiation. In this review, Section 1 briefly covers general background information on processes that control bone development as well as the biogenesis and function of miRNAs. In Section 2, we discuss the importance of miRNAs in skeletal development based on findings from in vivo mouse models and human clinical reports. Section 3 focuses on describing more recent data from the last three years related to miRNA regulation of osteoblast differentiation in vitro. Some of these studies also involve utilization of an in vivo rodent model to study the effects of miRNA modulation in scenarios of osteoporosis, bone repair or ectopic bone formation. In Section 4, we provide some recent information from studies analyzing the potential of miRNA-mediated crosstalk in bone and how exosomes containing miRNAs from one bone cell may affect the differentiation or function of another bone cell type. We then conclude by summarizing where the field currently stands with respect to miRNA-mediated regulation of osteogenesis and how information gained from developmental processes can be instructive in identifying potential therapeutic miRNA targets for the treatment of certain bone conditions.

Long non-coding RNA GRASLND enhances chondrogenesis via suppression of the interferon type II signaling pathway.

The roles of long noncoding RNAs (lncRNAs) in musculoskeletal development, disease, and regeneration remain poorly understood. Here, we identified the novel lncRNA GRASLND (originally named RNF144A-AS1) as a regulator of mesenchymal stem cell (MSC) chondrogenesis. GRASLND, a primate-specific lncRNA, is upregulated during MSC chondrogenesis and appears to act directly downstream of SOX9, but not TGF-ß3. We showed that the silencing of GRASLND resulted in lower accumulation of cartilage-like extracellular matrix in a pellet assay, while GRASLND overexpression - either via transgene ectopic expression or by endogenous activation via CRISPR-dCas9-VP64 - significantly enhanced cartilage matrix production. GRASLND acts to inhibit IFN-γ by binding to EIF2AK2, and we further demonstrated that GRASLND exhibits a protective effect in engineered cartilage against interferon type II. Our results indicate an important role of GRASLND in regulating stem cell chondrogenesis, as well as its therapeutic potential in the treatment of cartilage-related diseases, such as osteoarthritis.

Inhibition of 4-aminobutyrate aminotransferase protects against injury-induced osteoarthritis in mice.

Recently we demonstrated that ablation of the DNA methyltransferase enzyme, Dnmt3b, resulted in catabolism and progression of osteoarthritis (OA) in murine articular cartilage through a mechanism involving increased mitochondrial respiration. In this study, we identify 4-aminobutyrate aminotransferase (Abat) as a downstream target of Dnmt3b. Abat is an enzyme that metabolizes γ-aminobutyric acid to succinate, a key intermediate in the tricarboxylic acid cycle. We show that Dnmt3b binds to the Abat promoter, increases methylation of a conserved CpG sequence just upstream of the transcriptional start site, and inhibits Abat expression. Dnmt3b deletion in articular chondrocytes results in reduced methylation of the CpG sequence in the Abat promoter, which subsequently increases expression of Abat. Increased Abat expression in chondrocytes leads to enhanced mitochondrial respiration and elevated expression of catabolic genes. Overexpression of Abat in murine knee joints via lentiviral injection results in accelerated cartilage degradation following surgical induction of OA. In contrast, lentiviral-based knockdown of Abat attenuates the expression of IL-1ß-induced catabolic genes in primary murine articular chondrocytes in vitro and also protects against murine articular cartilage degradation in vivo. Strikingly, treatment with the FDA-approved small-molecule Abat inhibitor, vigabatrin, significantly prevents the development of injury-induced OA in mice. In summary, these studies establish Abat as an important new target for therapies to prevent OA.

MicroRNA-181a/b-1 over-expression enhances osteogenesis by modulating PTEN/PI3K/AKT signaling and mitochondrial metabolism.

MicroRNAs are small non-coding RNAs that play important roles in many cellular processes including proliferation, metabolism and differentiation. They function by binding to specific regions within the 3'UTR of target mRNAs resulting in suppression of protein synthesis and modulation of potentially many cellular pathways. We previously showed that miRNA expression levels differed between cells from distinct regions of developing human embryonic long bones. Specifically, we found that miR-181a-1 was significantly more highly expressed in hypertrophic chondrocytes compared to proliferating differentiated or progenitor chondrocytes, suggesting a potential role in regulating chondrocyte hypertrophy and/or endochondral bone formation. The goal of this study was to determine how miR-181a-1 together with its clustered miRNA, miR-181b-1, regulates osteogenesis. We show that over-expression of the miR-181a/b-1 cluster enhanced osteogenesis and that cellular pathways associated with protein synthesis and mitochondrial metabolism were significantly up-regulated. Metabolic assays revealed that the oxygen consumption rate and ATP-linked respiration were increased by miR-181a/b-1. To further decipher a potential mechanism causing these metabolic changes, we showed that PTEN (phosphatase and tensin homolog) levels were suppressed following miR-181a/b-1 over-expression, and that PI3K/AKT signaling was subsequently increased. Over-expression of PTEN was found to attenuate the enhancing effects of miR-181a/b-1, providing further evidence that miR-181a/b-1 regulates the PTEN/PI3K/AKT axis to enhance osteogenic differentiation and mitochondrial metabolism. These findings have important implications for the design of miR-181a/b targeting strategies to treat bone conditions such as fractures or heterotopic ossification.

MicroRNA-138 Inhibits Osteogenic Differentiation and Mineralization of Human Dedifferentiated Chondrocytes by Regulating RhoC and the Actin Cytoskeleton.

MicroRNAs (miRNAs) are known to play critical roles in many cellular processes including those regulating skeletal development and homeostasis. A previous study from our group identified differentially expressed miRNAs in the developing human growth plate. Among those more highly expressed in hypertrophic chondrocytes compared to progenitor chondrocytes was miR-138, therefore suggesting a possible role for this miRNA in regulating chondrogenesis and/or endochondral ossification. The goal of this study was to determine the function of miR- 138 in regulating osteogenesis by using human osteoarthritic dedifferentiated chondrocytes (DDCs) as source of inducible cells. We show that over-expression of miR-138 inhibited osteogenic differentiation of DDCs in vitro. Moreover, cell shape was altered and cell proliferation and possibly migration was also suppressed by miR-138. Given alterations in cell shape, closer analysis revealed that F-actin polymerization was also inhibited by miR-138. Computational approaches showed that the small GTPase, RhoC, is a potential miR-138 target gene. We pursued RhoC further given its function in regulating cell proliferation and migration in cancer cells. Indeed, miR-138 over-expression in DDCs resulted in decreased RhoC protein levels. A series of rescue experiments showed that RhoC over-expression could attenuate the inhibitory actions of miR-138 on DDC proliferation, F-actin polymerization and osteogenic differentiation. Bone formation was also found to be enhanced within human demineralized bone scaffolds seeded with DDCs expressing both miR-138 and RhoC. In conclusion, we have discovered a new mechanism in DDCs whereby miR-138 functions to suppress RhoC which subsequently inhibits proliferation, F-actin polymerization and osteogenic differentiation. To date, there are no published reports on the importance of RhoC in regulating osteogenesis. This opens up new avenues of research involving miR-138 and RhoC pathways to better understand mechanisms regulating bone formation in addition to the potential use of DDCs as a cell source for bone tissue engineering. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

MicroRNAs in orthopaedic research: Disease associations, potential therapeutic applications, and perspectives.

MicroRNAs (miRNAs) are small non-coding RNAs that function to control many cellular processes by their ability to suppress expression of specific target genes. Tens to hundreds of target genes may be affected by one miRNA, thereby resulting in modulation of multiple pathways in any given cell type. Therefore, altered expression of miRNAs (i.e., during tissue development or in scenarios of disease or cellular stress) can have a profound impact on processes regulating cell differentiation, metabolism, proliferation, or apoptosis, for example. Over the past 5-10 years, thousands of reports have been published on miRNAs in cartilage and bone biology or disease, thus highlighting the significance of these non-coding RNAs in regulating skeletal development and homeostasis. For the purpose of this review, we will focus on miRNAs or miRNA families that have demonstrated function in vivo within the context of cartilage, bone or other orthopaedic-related tissues (excluding muscle). Specifically, we will discuss studies that have utilized miRNA transgenic mouse models or in vivo approaches to target a miRNA with the aim of altering conditions such as osteoarthritis, osteoporosis and bone fractures in rodents. We will not discuss miRNAs in the context skeletal cancers since this topic is worthy of a review of its own. Overall, we aim to provide a comprehensive description of where the field currently stands with respect to the therapeutic potential of specific miRNAs to treat orthopaedic conditions and current technologies to target and modify miRNA function in vivo. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:33-51, 2018.