BACKGROUND

Osteoarthritis is a multifactorial disease characterized by destruction of the articular cartilage due to genetic, mechanical and environmental components affecting more than 100 million individuals all over the world. Despite the high prevalence of the disease, the absence of large-scale molecular studies limits our ability to understand the molecular pathobiology of osteoathritis and identify targets for drug development.

RESULTS

In this study we integrated genetic, bioinformatic and proteomic approaches in order to identify new genes and their collaborative networks involved in osteoarthritis pathogenesis. MicroRNA profiling of patient-derived osteoarthritic cartilage in comparison to normal cartilage, revealed a 16 microRNA osteoarthritis gene signature. Using reverse-phase protein arrays in the same tissues we detected 76 differentially expressed proteins between osteoarthritic and normal chondrocytes. Proteins such as SOX11, FGF23, KLF6, WWOX and GDF15 not implicated previously in the genesis of osteoarthritis were identified. Integration of microRNA and proteomic data with microRNA gene-target prediction algorithms, generated a potential "interactome" network consisting of 11 microRNAs and 58 proteins linked by 414 potential functional associations. Comparison of the molecular and clinical data, revealed specific microRNAs (miR-22, miR-103) and proteins (PPARA, BMP7, IL1B) to be highly correlated with Body Mass Index (BMI). Experimental validation revealed that miR-22 regulated PPARA and BMP7 expression and its inhibition blocked inflammatory and catabolic changes in osteoarthritic chondrocytes.

CONCLUSIONS

Our findings indicate that obesity and inflammation are related to osteoarthritis, a metabolic disease affected by microRNA deregulation. Gene network approaches provide new insights for elucidating the complexity of diseases such as osteoarthritis. The integration of microRNA, proteomic and clinical data provides a detailed picture of how a network state is correlated with disease and furthermore leads to the development of new treatments. This strategy will help to improve the understanding of the pathogenesis of multifactorial diseases such as osteoarthritis and provide possible novel therapeutic targets.

For many genes, ray-finned fish (Actinopterygii) have two paralogous copies, where only one ortholog is present in tetrapods. The discovery of an additional, almost-complete set of Hox clusters in teleosts (zebrafish, pufferfish, medaka, and cichlid) but not in basal actinopterygian lineages ( Polypterus) led to the formulation of the fish-specific genome duplication hypothesis. The phylogenetic timing of this genome duplication during the evolution of ray-finned fish is unknown, since only a few species of basal fish lineages have been investigated so far. In this study, three nuclear genes ( fzd8, sox11, tyrosinase) were sequenced from sturgeons (Acipenseriformes), gars (Semionotiformes), bony tongues (Osteoglossomorpha), and a tenpounder (Elopomorpha). For these three genes, two copies have been described previously teleosts (e.g., zebrafish, pufferfish), but only one orthologous copy is found in tetrapods. Individual gene trees for these three genes and a concatenated dataset support the hypothesis that the fish-specific genome duplication event took place after the split of the Acipenseriformes and the Semionotiformes from the lineage leading to teleost fish but before the divergence of Osteoglossiformes. If these three genes were duplicated during the proposed fish-specific genome duplication event, then this event separates the species-poor early-branching lineages from the species-rich teleost lineage. The additional number of genes resulting from this event might have facilitated the evolutionary radiation and the phenotypic diversification of the teleost fish.

The progression of neurogenesis relies on proneural basic helix-loop-helix (bHLH) transcription factors. These factors operate in undifferentiated neural stem cells and induce cell cycle exit and the initiation of a neurogenic program. However, the transient expression of proneural bHLH proteins in neural progenitors indicates that expression of neuronal traits must rely on previously unexplored mechanisms operating downstream from proneural bHLH proteins. Here we show that the HMG-box transcription factors Sox4 and Sox11 are of critical importance, downstream from proneural bHLH proteins, for the establishment of pan-neuronal protein expression. Examination of a neuronal gene promoter reveals that Sox4 and Sox11 exert their functions as transcriptional activators. Interestingly, the capacity of Sox4 and Sox11 to induce the expression of neuronal traits is independent of mechanisms regulating the exit of neural progenitors from the cell cycle. The transcriptional repressor protein REST/NRSF has been demonstrated to block neuronal gene expression in undifferentiated neural cells. We now show that REST/NRSF restricts the expression of Sox4 and Sox11, explaining how REST/NRSF can prevent precocious expression of neuronal proteins. Together, these findings demonstrate a central regulatory role of Sox4 and Sox11 during neuronal maturation and mechanistically separate cell cycle withdrawal from the establishment of neuronal properties.

Pluripotent embryonic stem (ES) cells can generate all cell types, but how cell lineages are initially specified and maintained during development remains largely unknown. Different classes of Sox transcription factors are expressed during neurogenesis and have been assigned important roles from early lineage specification to neuronal differentiation. Here we characterize the genome-wide binding for Sox2, Sox3, and Sox11, which have vital functions in ES cells, neural precursor cells (NPCs), and maturing neurons, respectively. The data demonstrate that Sox factor binding depends on developmental stage-specific constraints and reveal a remarkable sequential binding of Sox proteins to a common set of neural genes. Interestingly, in ES cells, Sox2 preselects for neural lineage-specific genes destined to be bound and activated by Sox3 in NPCs. In NPCs, Sox3 binds genes that are later bound and activated by Sox11 in differentiating neurons. Genes prebound by Sox proteins are associated with a bivalent chromatin signature, which is resolved into a permissive monovalent state upon binding of activating Sox factors. These data indicate that a single key transcription factor family acts sequentially to coordinate neural gene expression from the early lineage specification in pluripotent cells to later stages of neuronal development.

The high-mobility-group domain-containing transcription factor Sox11 is expressed transiently during embryonic development in many tissues that undergo inductive remodeling. Here we have analyzed the function of Sox11 by gene deletion in the mouse. Sox11-deficient mice died at birth from congenital cyanosis, likely resulting from heart defects. These included ventricular septation defects and outflow tract malformations that ranged from arterial common trunk to a condition known as double outlet right ventricle. Many other organs that normally express Sox11 also exhibited severe developmental defects. We observed various craniofacial and skeletal malformations, asplenia, and hypoplasia of the lung, stomach, and pancreas. Eyelids and the abdominal wall did not close properly in some Sox11-deficient mice. This phenotype suggests a prime function for Sox11 in tissue remodeling and identifies SOX11 as a potentially mutated gene in corresponding human malformation syndromes.

Intermediate-filament Nestin and group B1 SOX transcription factors (SOX1/2/3) are often employed as markers for neural primordium, suggesting their regulatory link. We have identified adjacent and essential SOX and POU factor binding sites in the Nestin neural enhancer. The 30-bp sequence of the enhancer including these sites (Nes30) showed a nervous system-specific and SOX-POU-dependent enhancer activity in multimeric forms in transfection assays and was utilized in assessing the specificity of the synergism; combinations of either group B1 or group C SOX (SOX11) with class III POU proved effective. In embryonic day 13.5 mouse spinal cord, Nestin was expressed in the cells with nuclei in the ventricular and subventricular zones. SOX1/2/3 expression was confined to the nuclei of the ventricular zone; SOX11 localized to the nuclei of both subventricular (high-level expression) and intermediate (low-level expression) zones. Class III POU (Brn2) was expressed at high levels, localizing to the nucleus in the ventricular and subventricular zones; moderate expression was observed in the intermediate zone, distributed in the cytoplasm. These data support the model that synergic interactions between group B1/C SOX and class III POU within the nucleus determine Nestin expression. Evidence also suggests that such interactions are involved in the regulation of neural primordial cells.

Cell fate can be reprogrammed by modifying intrinsic and extrinsic cues. Here we show that two small molecules (forskolin and dorsomorphin) enable the transcription factor Neurogenin 2 (NGN2) to convert human fetal lung fibroblasts into cholinergic neurons with high purity (>90%) and efficiency (up to 99% of NGN2-expressing cells). The conversion is direct without passing through a proliferative progenitor state. These human induced cholinergic neurons (hiCN) show mature electrophysiological properties and exhibit motor neuron-like features, including morphology, gene expression and the formation of functional neuromuscular junctions. Inclusion of an additional transcription factor, SOX11, also efficiently converts postnatal and adult skin fibroblasts from healthy and diseased human patients to cholinergic neurons. Taken together, this study identifies a simple and highly efficient strategy for reprogramming human fibroblasts to subtype-specific neurons. These findings offer a unique venue for investigating the molecular mechanisms underlying cellular plasticity and human neurodegenerative diseases.

Three chicken Sox (SRY-like box) genes have been identified that show an interactive pattern of expression in the developing embryonic nervous system. cSox2 and cSox3 code for related proteins and both are predominantly expressed in the immature neural epithelium of the entire CNS of HH stage 10 to 34 embryos. cSox11 is related to cSox2 and cSox3 only by virtue of containing an SRY-like HMG-box sequence but shows extensive homology with Sox-4 at its C-terminus. cSox11 is expressed in the neural epithelium but is transiently upregulated in maturing neurons after they leave the neural epithelium. These patterns of expression suggest that Sox genes play a role in neural development and that the developmental programme from immature to mature neurons may involve switching of Sox gene expression. cSox11 also exhibits a lineage restricted pattern of expression in the peripheral nervous system.

Mantle cell lymphoma (MCL) is typically a very aggressive disease with poor outcomes, but some cases display an indolent behavior that might not necessitate treatment at diagnosis. To define molecular criteria that might permit recognition of such cases, we compared the clinicopathologic features, gene expression, and genomic profile of patients who had indolent or conventional disease (iMCL or cMCL). Patients with iMCL displayed nonnodal leukemic disease with predominantly hypermutated IGVH and noncomplex karyotypes. iMCL and cMCL shared a common gene expression profile that differed from other leukemic lymphoid neoplasms. However, we identified a signature of 13 genes that was highly expressed in cMCL but underexpressed in iMCL. SOX11 was notable in this signature and we confirmed a restriction of SOX11 protein expression to cMCL. To validate the potential use of SOX11 as a biomarker for cMCL, we evaluated SOX11 protein expression in an independent series of 112 cases of MCL. Fifteen patients with SOX11-negative tumors exhibited more frequent nonnodal presentation and better survival compared with 97 patients with SOX11-positive MCL (5-year overall survival of 78% versus 36%, respectively; P = 0.001). In conclusion, we defined nonnodal presentation, predominantly hypermutated IGVH, lack of genomic complexity, and absence of SOX11 expression as qualities of a specific subtype of iMCL with excellent outcomes that might be managed more conservatively than cMCL.

The group C of Sry-related high-mobility group (HMG) box (Sox) transcription factors has three members in most vertebrates: Sox4, Sox11 and Sox12. Sox4 and Sox11 have key roles in cardiac, neuronal and other major developmental processes, but their molecular roles in many lineages and the roles of Sox12 remain largely unknown. We show here that the three genes are co-expressed at high levels in neuronal and mesenchymal tissues in the developing mouse, and at variable relative levels in many other tissues. The three proteins have conserved remarkable identity through evolution in the HMG box DNA-binding domain and in the C-terminal 33 residues, and we demonstrate that the latter residues constitute their transactivation domain (TAD). Sox11 activates transcription several times more efficiently than Sox4 and up to one order of magnitude more efficiently than Sox12, owing to a more stable alpha-helical structure of its TAD. This domain and acidic domains interfere with DNA binding, Sox11 being most affected and Sox4 least affected. The proteins are nevertheless capable of competing with one another in reporter gene transactivation. We conclude that the three SoxC proteins have conserved overlapping expression patterns and molecular properties, and might therefore act in concert to fulfill essential roles in vivo.

During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. In this study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway.

BACKGROUND

Cyclin D1-negative mantle cell lymphoma is difficult to distinguish from other small B-cell lymphomas. The clinical and pathological characteristics of patients with this form of lymphoma have not been well defined. Overexpression of the transcription factor SOX11 has been observed in conventional mantle cell lymphoma. The aim of this study was to determine whether this gene is expressed in cyclin D1-negative mantle cell lymphoma and whether its detection may be useful to identify these tumors.

METHODS

The microarray database of 238 mature B-cell neoplasms was re-examined. SOX11 protein expression was investigated immunohistochemically in 12 cases of cyclin D1-negative mantle cell lymphoma, 54 cases of conventional mantle cell lymphoma, and 209 additional lymphoid neoplasms.

RESULTS

SOX11 mRNA was highly expressed in conventional and cyclin D1-negative mantle cell lymphoma and in 33% of the cases of Burkitt's lymphoma but not in any other mature lymphoid neoplasm. SOX11 nuclear protein was detected in 50 cases (93%) of conventional mantle cell lymphoma and also in the 12 cyclin D1-negative cases of mantle cell lymphoma, the six cases of lymphoblastic lymphomas, in two of eight cases of Burkitt's lymphoma, and in two of three T-prolymphocytic leukemias but was negative in the remaining lymphoid neoplasms. Cyclin D2 and D3 mRNA levels were significantly higher in cyclin D1-negative mantle cell lymphoma than in conventional mantle cell lymphoma but the protein expression was not discriminative. The clinico-pathological features and outcomes of the patients with cyclin D1-negative mantle cell lymphoma identified by SOX11 expression were similar to those of patients with conventional mantle cell lymphoma.

CONCLUSIONS

SOX11 mRNA and nuclear protein expression is a highly specific marker for both cyclin D1-positive and negative mantle cell lymphoma.

Glial cells of the oligodendrocyte lineage express several highly related POU proteins including Tst-1/Oct6/SCIP and Brn-1. Tst-1/Oct6/SCIP, but not Brn-1 efficiently cooperated with Sox10, the only SRY box protein so far identified in oligodendrocytes. Here we show that, in addition to Sox10, cells of the oligodendrocyte lineage contain significant amounts of the related SRY box proteins Sox4 and Sox11. During development, Sox11 was strongly expressed in the central nervous system. It was first detected in neural precursors throughout the neuroepithelium. During later stages of neural development, Sox11 was additionally expressed in areas of the brain in which neurons undergo differentiation. In agreement with its expression in neural precursors, Sox11 levels in cells of the oligodendrocyte lineage were high in precursors and down-regulated during terminal differentiation. Outside the nervous system, expression of Sox11 was also detected in the developing limbs, face, and kidneys. Structure function analysis revealed that Sox11 has a strong intrinsic transactivation capacity which is mediated by a transactivation domain in its carboxyl-terminal part. In addition, Sox11 efficiently synergized with Brn-1. Synergy was dependent on binding of both proteins to adjacent DNA elements, and required the presence of the respective transactivation domain in each protein. Our data suggest the existence of a specific code in which POU proteins require specific Sox proteins to exhibit cooperative effects in glial cells.

Numerous transcriptional regulators of neurogenesis have been identified in the developing and adult brain, but how neurogenic fate is programmed at the epigenetic level remains poorly defined. Here, we report that the transcription factor Pax6 directly interacts with the Brg1-containing BAF complex in adult neural progenitors. Deletion of either Brg1 or Pax6 in the subependymal zone (SEZ) causes the progeny of adult neural stem cells to convert to the ependymal lineage within the SEZ while migrating neuroblasts convert to different glial lineages en route to or in the olfactory bulb (OB). Genome-wide analyses reveal that the majority of genes downregulated in the Brg1 null SEZ and OB contain Pax6 binding sites and are also downregulated in Pax6 null SEZ and OB. Downstream of the Pax6-BAF complex, we find that Sox11, Nfib, and Pou3f4 form a transcriptional cross-regulatory network that drives neurogenesis and can convert postnatal glia into neurons. Taken together, elements of our work identify a tripartite effector network activated by Pax6-BAF that programs neuronal fate.

The co-emergence of a six-layered cerebral neocortex and its corticospinal output system is one of the evolutionary hallmarks of mammals. However, the genetic programs that underlie their development and evolution remain poorly understood. Here we identify a conserved non-exonic element (E4) that acts as a cortex-specific enhancer for the nearby gene Fezf2 (also known as Fezl and Zfp312), which is required for the specification of corticospinal neuron identity and connectivity. We find that SOX4 and SOX11 functionally compete with the repressor SOX5 in the transactivation of E4. Cortex-specific double deletion of Sox4 and Sox11 leads to the loss of Fezf2 expression, failed specification of corticospinal neurons and, independent of Fezf2, a reeler-like inversion of layers. We show evidence supporting the emergence of functional SOX-binding sites in E4 during tetrapod evolution, and their subsequent stabilization in mammals and possibly amniotes. These findings reveal that SOX transcription factors converge onto a cis-acting element of Fezf2 and form critical components of a regulatory network controlling the identity and connectivity of corticospinal neurons.

Among the families of transcription factors expressed at the neural plate border in response to neural crest-inducing signals, Sox proteins have emerged as important players in regulating multiple aspects of neural crest development. Here, we summarize the expression of six Sox genes, namely Sox8, Sox9, Sox10, LSox5, Sox4 and Sox11, in neural crest progenitors and their derivatives, and review some aspects of their function pertaining to neural crest development in several species.

For successful nerve regeneration, a coordinated shift in gene expression pattern must occur in axotomized neurons. To identify genes participating in axonal regeneration, we characterized mRNA expression profiles in dorsal root ganglia (DRG) before and after sciatic nerve transection. Dozens of genes are differentially expressed after sciatic nerve injury by microarray analysis. Induction of SOX11, FLRT3, myosin-X, and fibroblast growth factor-inducible-14 (Fn14) mRNA in axotomized DRG neurons was verified by Northern analysis and in situ hybridization. The Fn14 gene encodes a tumor necrosis-like weak inducer of apoptosis (TWEAK) receptor and is dramatically induced in DRG neurons after nerve damage, despite low expression in developing DRG neurons. Fn14 expression in PC12 cells is also upregulated by nerve growth factor treatment. Overexpression of Fn14 promotes growth cone lamelipodial formation and increases neurite outgrowth in PC12 cells. These Fn14 effects are independent of the ligand, TWEAK. Fn14 colocalizes with the Rho family GTPases, Cdc42 and Rac1. Furthermore, Fn14 physically associates with Rac1 GTPase in immunoprecipitation studies. The neurite outgrowth-promoting effect of Fn14 is enhanced by Rac1 activation and suppressed by Rac1 inactivation. These findings suggest that Fn14 contributes to nerve regeneration via a Rac1 GTPase-dependent mechanism.

The transcription factor Sox11 is expressed at high levels in developing sensory neurons and injured adult neurons but little is known about its transcriptional targets and function. In this study we examined the role of Sox11 using Neuro2a neuroblastoma cells and cultured mouse dorsal root ganglia (DRG) neurons. Results show Sox11 has an essential role in regulation of neuron survival and neurite outgrowth in Neuro2a cells and primary sensory neurons. Neuro2a cells increase expression of Sox11 as they differentiate in culture. Following addition of 20 microM retinoic acid (RA), a stimulus for differentiation that enhances neurite growth and differentiation, Sox11 level rises. RNAi-mediated knockdown of Sox11 in RA-differentiated Neuro2a cells caused a decrease in neurite growth and an increase in the percent of apoptotic cells. RNA expression analysis showed that Sox11 knockdown modulated the level of mRNAs encoding several genes related to cell survival and death. Further validation in the Neuro2a model showed Sox11 knockdown increased expression of the pro-apoptotic gene BNIP3 (BclII interacting protein 1 NIP3) and decreased expression of the anti-apoptotic gene TANK (TNF receptor-associated factor family member-associated NFkappaB activator). Cultured primary DRG neurons also express Sox11 and treatment with Sox11 small interfering RNA (siRNA) caused a significant decrease in neurite growth and branching and a decrease in mRNA encoding actin-related protein complex 3 (Arpc3), an actin organizing protein that may be involved in axon growth. The percent of apoptotic neurons also increased in cultures of DRG neurons treated with Sox11 siRNA. Similar to Neuro2a cells, a decrease in TANK gene expression occurred, suggesting at least some overlap in Sox11 transcriptional targets in Neuro2a and DRG neurons. These data are consistent with a central role for Sox11 in regulating events that promote neurite growth and neuron survival.

LINEs are endogenous mobile genetic elements which have dispersed and accumulated in the genomes of most higher eukaryotes via germline transposition, with up to 100 000 copies for the human LINE-1 (L1H) sequences. Although severely repressed in most normal tissues, L1H is still functional, with evidence for both germline and somatic-essentially in tumors-transpositions. Yet, no transcription factor that could regulate their transcription and be responsible for their transposition has hitherto been described. Here we show that factors belonging to the family of the testis-determining factor gene SRY (the SOX family) can modulate L1H promoter activity over a 10-fold range in a transient transfection assay using a luciferase reporter gene. These effects depend on two functional SRY binding sites which can be identified within the L1H promoter via mobility shift assays. Induction of endogenous L1Hs upon ectopic expression of the SOX11 transcription factor is further demonstrated, thus strengthening the physiological relevance of these new-and highly dispersed-target sites for the otherwise unclassical transcription factors of the SRY family.

The transcription factor-encoding gene, Sox4, is expressed in a wide range of tissues and has been shown to be functionally involved in heart, B-cell and reproductive system development. Sox4 shows a high degree of sequence homology with another group C Sox gene, Sox11, which is predominantly expressed in the CNS. Since the expression of Sox4 in the CNS has not been described we have carried out such a study. Sox4 and Sox11 expression increased simultaneously in the same early differentiating cells of the developing CNS except in the external granule layer of the cerebellum where Sox11 expression preceded that of Sox4. As development proceeded, their expression always appeared to relate to the maturational stage of the cell population, with Sox11 expression more transient than Sox4, except in the spinal cord where the reverse was true. Sox4 knock-out mice have been shown to die of a heart defect half way through gestation with no observable CNS phenotype. Our more detailed analysis showed no abnormality in the spatial restriction of expression of Sox2, Sox11, Mash1, neurogenin1 or neurogenin2, although the level of expression of Sox11 and Mash1 appeared a little different from the wild-type, implying that Sox4 might indeed have a functional role in CNS development. However, since Sox4 and Sox11 expression is so similar, we propose that Sox11 might compensate for the loss of Sox4 function in the CNS such that the phenotype is extremely mild in the Sox4 null mutant.

Sox4, Sox11 and Sox12 constitute the group C of Sry-related HMG box proteins. They are co-expressed in embryonic neuronal progenitors and in mesenchymal cells in many developing organs. More closely related to each other than to any other proteins, they nevertheless bind DNA and activate transcription in vitro with different efficiencies. Sox4-null embryos and Sox11-null newborns die from heart malformations and the latter display widespread defects, while Sox12-null mice are viable and do not show obvious malformations. Sox4 facilitates differentiation of lymphocytes, pancreatic beta cells, osteoblasts and acts in redundancy with Sox11 to promote neuronal differentiation. Sox4 and Sox11 are upregulated in many tumor types in humans, where their roles in cell survival, proliferation, and metastasis remain controversial. Together, these data hint that Sox4 and Sox11 regulate cell differentiation, proliferation and survival in multiple essential processes, and suggest that they may act in redundancy to control many more developmental, physiological and pathological processes than currently known.