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.

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.

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.

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.

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.

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.

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.

miR-483 targets SMAD4 to suppress chondrogenic differentiation of human mesenchymal stem cells.

MicroRNAs (miRNAs) can regulate cellular differentiation processes by modulating multiple pathways simultaneously. Previous studies to analyze in vivo miRNA expression patterns in developing human limb cartilage tissue identified significant downregulation of miR-483 in hypertrophic chondrocytes relative to proliferating and differentiated chondrocytes. To test the function of miR-483 during chondrogenesis, lentiviral strategies were used to overexpress miR-483 during in vitro chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). While the in vivo expression patterns led us to hypothesize that miR-483 may enhance chondrogenesis or suppress hypertrophic marker expression, surprisingly, miR-483 overexpression reduced chondrocyte gene expression and cartilage matrix production. In addition, cell death was induced at later stages of the chondrogenesis assay. Mechanistic studies revealed that miR-483 overexpression resulted in downregulation of the TGF-ß pathway member SMAD4, a known direct target of miR-483-3p. From these studies, we conclude that constitutive overexpression of miR-483 in hBM-MSCs inhibits chondrogenesis of these cells and does not represent an effective strategy to attempt to enhance chondrocyte differentiation and anabolism in this system in vitro. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2369-2377, 2017.

Emerging roles for long noncoding RNAs in skeletal biology and disease.

Normal skeletal development requires tight coordination of transcriptional networks, signaling pathways, and biomechanical cues, and many of these pathways are dysregulated in pathological conditions affecting cartilage and bone. Recently, a significant role has been identified for long noncoding RNAs (lncRNAs) in developing and maintaining cellular phenotypes, and improvements in sequencing technologies have led to the identification of thousands of lncRNAs across diverse cell types, including the cells within cartilage and bone. It is clear that lncRNAs play critical roles in regulating gene expression. For example, they can function as epigenetic regulators in the nucleus via chromatin modulation to control gene transcription, or in the cytoplasm, where they can function as scaffolds for protein-binding partners or modulate the activity of other coding and noncoding RNAs. In this review, we discuss the growing list of lncRNAs involved in normal development and/or homeostasis of the skeletal system, the potential mechanisms by which these lncRNAs might function, and recent improvements in the methodologies available to study lncRNA functions in vitro and in vivo. Finally, we address the likely utility of lncRNAs as biomarkers and therapeutic targets for diseases of the skeletal system, including osteoarthritis, osteoporosis, and in cancers of the skeletal system.

Hypoxia-inducible factor 3-alpha expression is associated with the stable chondrocyte phenotype.

The hypoxia-inducible factors HIF-1α and HIF-2α are important regulators of the chondrocyte phenotype but little is known about HIF-3α in cartilage. The objective of this study was to characterize HIF-3α (HIF3A) expression during chondrocyte differentiation in vitro and in native cartilage tissues. HIF3A, COL10A1, and MMP13 were quantified in mesenchymal stem cells (MSCs) and articular chondrocytes from healthy and osteoarthritic (OA) tissue in three-dimensional cultures and in human embryonic epiphyses and adult articular cartilage. HIF3A was found to have an inverse association with hypertrophic markers COL10A1 and MMP13 in chondrogenic cells and tissues. In healthy chondrocytes, HIF3A was induced by dexamethasone and increased during redifferentiation. By comparison, HIF3A expression was extremely low in chondrogenically differentiated MSCs expressing high levels of COL10A1 and MMP13. HIF3A was also lower in redifferentiated OA chondrocytes than in healthy chondrocytes. In human embryonic epiphyseal tissue, HIF3A expression was lowest in the hypertrophic zone. Distinct splice patterns were also found in embryonic cartilage when compared with adult articular cartilage and redifferentiated chondrocytes. These in vitro and in vivo findings suggest that HIF3A levels are indicative of the hypertrophic state of chondrogenic cells and one or more splice variants may be important regulators of the chondrocyte phenotype.

Changes in type II procollagen isoform expression during chondrogenesis by disruption of an alternative 5' splice site within Col2a1 exon 2.

This study describes a new mechanism controlling the production of alternatively spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codons in exon 6 (type IIC). This transcript is produced by utilization of another 5' splice site present in exon 2. To determine the role of this IIC splicing event in vivo, we generated transgenic mice containing silent knock-in mutations at the IIC 5' splice site (Col2a1-mIIC), thereby inhibiting production of IIC transcripts. Heterozygous and homozygous knock-in mice were viable and display no overt skeletal phenotype to date. However, RNA expression profiles revealed that chondrocytes in cartilage from an age range of Col2a1-mIIC mice produced higher levels of IIA and IID mRNAs and decreased levels of IIB mRNAs throughout pre-natal and post-natal development, when compared to chondrocytes from littermate control mice. Immunofluorescence analyses showed a clear increase in expression of embryonic type II collagen protein isoforms (i.e. containing the exon 2-encoded cysteine-rich (CR) protein domain) in cartilage extracellular matrix (ECM). Interestingly, at P14, P28 and P56, expression of embryonic Col2a1 isoforms in Col2a1-mIIC mice persisted in the pericellular domain of the ECM in articular and growth plate cartilage. We also show that persistent expression of the exon 2-encoded CR domain in the ECM of post-natal cartilage tissue may be due, in part, to the embryonic form of type XI collagen (the α3 chain of which is also encoded by the Col2a1 gene). In conclusion, expression of the Col2a1 IIC splice form may have a regulatory function in controlling alternative splicing of exon 2 to generate defined proportions of IIA, IID and IIB procollagen isoforms during cartilage development. Future studies will involve ultrastructural and biomechanical analysis of the collagen ECM to determine the effects of persistent mis-expression of embryonic collagen isoforms in mature cartilage tissue.

Quantification of type II procollagen splice forms using alternative transcript-qPCR (AT-qPCR).

During skeletal development, the onset of chondrogenic differentiation is marked by expression of the α1(II) procollagen (Col2a1) gene. Exon 2 of Col2a1 codes for a cysteine-rich von Willebrand factor C-like domain. Chondroprogenitors express the exon 2-containing IIA and IID splice forms by utilizing adjacent 5' splice sites separated by 3 base pairs. There is a shift to expression of the shorter, exon 2-lacking IIB splice form with further differentiation. Alternative splicing analysis of Col2a1 splice forms has often relied upon semi-quantitative PCR, using a single set of PCR primers to amplify multiple splice forms. We show that this widely used method is inaccurate due to mismatched amplification efficiency of different-sized PCR products. We have developed the TaqMan®-based AT-qPCR (Alternative Transcript-qPCR) assay to more accurately quantify alternatively spliced mRNA, and demonstrate the measurement of Col2a1 splice form expression in differentiating ATDC5 cells in vitro, and in wild type mouse embryonic and postnatal cartilage in vivo. The AT-qPCR assay is based on the use of a multiple-amplicon standard (MAS) plasmid, containing a chemically synthesized cluster of splice site-spanning PCR amplicons, to quantify alternative splice forms by standard curve-based qPCR. The MAS plasmid designed for Col2a1 also contained an 18S rRNA amplicon for sample normalization, and an amplicon corresponding to a region spanning exon 52-53 to measure total Col2a1 mRNA. In mouse E12.5 to P70 cartilages, we observed the expected switch between the IIA and IIB splice forms; no IID or IIC splice products were observed. However, in the ATDC5 cultures, predominant expression of the IIA and IID splice forms was found at all times in culture. Additionally, we observed that the sum of the IIA, IIB and IID splice forms comprises only a small fraction of Col2a1 transcripts containing the constitutive exon 52-53 junction. We conclude from our results that the majority of ATDC5 cells in the assay described in this study remained as chondroprogenitors during culture in standard differentiation conditions, and that additional Col2a1 transcripts may be present. The validity of this novel AT-qPCR assay was confirmed by demonstrating the expected Col2a1 isoform expression patterns in vivo in developing mouse cartilage. The ability to measure true levels of procollagen type II splice forms will provide better monitoring of chondrocyte differentiation in other model systems. In addition, the AT-qPCR assay described here could be applied to any gene of interest to detect and quantify known and predicted alternative splice forms and can be scaled up for high throughput assays.

Constitutively activated NLRP3 inflammasome causes inflammation and abnormal skeletal development in mice.

The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1ß. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1ß and systemic inflammation are common to all cryopyrinopathy disorders, skeletal abnormalities, prominently in the knees, and low bone mass are unique features of patients with NOMID. To gain insights into the mechanisms underlying skeletal abnormalities in NOMID, we generated knock-in mice globally expressing the D301N NLRP3 mutation (ortholog of D303N in human NLRP3). NOMID mice exhibit neutrophilia in blood and many tissues, including knee joints, and high levels of serum inflammatory mediators. They also exhibit growth retardation and severe postnatal osteopenia stemming at least in part from abnormally accelerated bone resorption, attended by increased osteoclastogenesis. Histologic analysis of knee joints revealed abnormal growth plates, with loss of chondrocytes and growth arrest in the central region of the epiphyses. Most strikingly, a tissue "spike" was observed in the mid-region of the growth plate in the long bones of all NOMID mice that may be the precursor to more severe deformations analogous to those observed in NOMID patients. These findings provide direct evidence linking a NOMID-associated NLRP3-activating mutation to abnormalities of postnatal skeletal growth and bone remodeling.

Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model.

The present study describes the generation of a knock-in mouse model to address the role of type II procollagen (Col2a1) alternative splicing in skeletal development and maintenance. Alternative splicing of Col2a1 precursor mRNA is a developmentally-regulated event that only occurs in chondrogenic tissue. Normally, chondroprogenitor cells synthesize predominantly exon 2-containing mRNA isoforms (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. Another isoform, IIC, has also been identified that contains a truncated exon 2 and is not translated into protein. The biological significance of this IIA/IID to IIB splicing switch is not known. Utilizing a splice site targeting knock-in approach, a 4 nucleotide mutation was created to convert the 5' splice site of Col2a1 exon 2 from a weak, non-consensus sequence to a strong, consensus splice site. This resulted in apparent expression of only the IIA mRNA isoform, as confirmed in vitro by splicing of a type II procollagen mini-gene containing the 5' splice site mutation. To test the splice site targeting approach in vivo, homozygote mice engineered to retain IIA exon 2 (Col2a1(+ex2)) were generated. Chondrocytes from hindlimb epiphyseal cartilage of homozygote mice were shown to express only IIA mRNA and protein at all pre- and post-natal developmental stages analyzed (E12.5, E16.5, P0, P3, P7, P14, P28 and P70). As expected, type IIB procollagen was the major isoform produced in wild type cartilage at all post-natal time points. Col2a1(+ex2) homozygote mice are viable, appear healthy and display no overt phenotype to date. However, research is currently underway to investigate the biological consequence of persistent expression of the exon 2-encoded conserved cysteine-rich domain in post-natal skeletal tissues.

Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-ß3.

Biomaterial microparticles are commonly utilized as growth factor delivery vehicles to induce chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs). To address whether the presence of microparticles could themselves affect differentiation of MSCs, a 3D co-aggregate system was developed containing an equal volume of human primary bone marrow-derived MSCs and non-degradable RGD-conjugated poly(ethylene glycol) microspheres (PEG-µs). Following TGF-ß3 induction, differences in cell phenotype, gene expression and protein localization patterns were found when compared to MSC aggregate cultures devoid of PEG-µs. An outer fibrous layer always found in differentiated MSC aggregate cultures was not formed in the presence of PEG-µs. Type II collagen protein was synthesized by cells in both culture systems, although increased levels of the long (embryonic) procollagen isoforms were found in MSC/PEG-µs aggregates. Ubiquitous deposition of type I and type X collagen proteins was found in MSC/PEG-µs cultures while the expression patterns of these collagens was restricted to specific areas in MSC aggregates. These findings show that MSCs respond differently to TGF-ß3 when in a PEG-µs environment due to effects of cell dilution, altered growth factor diffusion and/or cellular interactions with the microspheres. Although not all of the expression patterns pointed toward improved chondrogenic differentiation in the MSC/PEG-µs cultures, the surprisingly large impact of the microparticles themselves should be considered when designing drug delivery/scaffold strategies.

Upregulation of Runx2 and Osterix during in vitro chondrogenesis of human adipose-derived stromal cells.

The aim of this study was to create a gene expression profile to better define the phenotype of human adipose-derived stromal cells (HADSCs) during in vitro chondrogenesis, osteogenesis and adipogenesis. A novel aspect of this work was the analysis of the same subset of genes during HADSC differentiation into all three lineages. Chondrogenic induction resulted in increased mRNA expression of Sox transcription factors, COL2A1,COL10A1, Runx2, and Osterix. This is the first report demonstrating significant upregulation in expression of osteogenesis-related transcription factors Runx2 and Osterix by TGF-beta3 induction of HADSCs during in vitro chondrogenesis. These findings suggest that the commonly-used chondrogenic induction reagents promote differentiation suggestive of hypertrophic chondrocytes and osteoblasts. We conclude that alternative strategies are required to induce efficient articular chondrocyte differentiation in order for HADSCs to be of clinical use in cartilage tissue engineering.

A novel tumor necrosis factor alpha-responsive CCAAT/enhancer binding protein site regulates expression of the cartilage-derived retinoic acid-sensitive protein gene in cartilage.

OBJECTIVE: Inflammatory processes in rheumatoid arthritis are primarily regulated by the cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta). Previous studies in our laboratory have shown that IL-1beta represses expression of the cartilage characteristic genes, cartilage-derived retinoic acid-sensitive protein (cd-rap) and type II collagen (COL2A1); this mechanism of repression involves activation of a CCAAT/enhancer binding protein (c/EBP) site within promoter regions. The aim of this study was to investigate novel TNFalpha-mediated mechanisms that regulate the expression of cd-rap. METHODS: Rat chondrosarcoma cells were transiently transfected with complementary DNA constructs encoding cd-rap, in the presence of TNFalpha. The expression of c/EBPbeta, SOX9, and p300 in rat chondrosarcoma cells and primary human articular chondrocytes after treatment with TNFalpha was examined by reverse transcription-polymerase chain reaction and Western blotting. The effect of TNFalpha on endogenous binding of c/EBPbeta or SOX9 to the cd-rap promoter was examined by chromatin immunoprecipitation assays. RESULTS: We identified a new c/EBP binding site in the cd-rap promoter (from position -1059 bp to position -1046 bp). Binding of c/EBP to this site was regulated by TNFalpha but not IL-1beta, resulting in down-regulation of cd-rap expression. This effect was reversed by mutational inactivation of the c/EBP motif. In addition, the activation potential of SOX9 and CREB binding protein/p300 on the cd-rap promoter was enhanced after mutation of the new c/EBP binding site, indicating that blockage of this site would increase transcription. CONCLUSION: TNFalpha regulates the expression and/or DNA-binding potential of key positive-acting and negative-acting transcription factors that control the expression of the cartilage matrix gene, cd-rap.

Expression of two novel alternatively spliced COL2A1 isoforms during chondrocyte differentiation.

Alternative splicing of the type II procollagen gene (COL2A1) is developmentally regulated during chondrogenesis. Type IIA procollagen (+ exon 2) is synthesized by chondroprogenitor cells while type IIB procollagen (- exon 2) is synthesized by differentiated chondrocytes. Here, we report expression of two additional alternatively spliced COL2A1 isoforms during chondrocyte differentiation of bone marrow-derived mesenchymal stem cells (MSCs). One isoform, named IIC, contains only the first 34 nucleotides of exon 2 by the use of an alternative 5' splice site, resulting in a premature termination codon and possible nonsense-mediated decay of IIC mRNA. Low levels of the IIC isoform were detected by RT-PCR and Southern analysis of COL2A1 cDNA amplified from differentiating rabbit and human MSCs. A second novel transcript, named IID, arises by the use of another 5' alternative splice site in intron 2. The IID isoform contains exon 2 plus 3 nucleotides, resulting in the insertion of an additional amino acid. The IID isoform was co-expressed with the IIA isoform during chondrogenesis, and was approximately one-third as abundant. Deletion of the IIC alternative 5' splice site from a COL2A1 mini-gene construct resulted in a significant increase in the IIA:IIB ratio. A mutant mini-gene that inhibited production of the IID isoform, however, had differential effects on the production of the IIA and IIB isoforms: this was apparently related to the differentiation status of the cell type used. These data suggest that COL2A1 mRNA abundance and other aspects of chondrocyte differentiation may be regulated by the use of these previously undetermined alternative splice sites.