Mesenchymal stem cell differentiation is controlled by the cooperative activity of a network of signaling mechanisms. Among these, RUNX2 and SOX9 are the major transcription factors for osteogenesis and chondrogenesis, respectively. Their expression is overlapped both temporally and spatially during embryogenesis. Here we have demonstrated that RUNX2 and SOX9 physically interact in intact cells and have confirmed that SOX9 can inhibit the transactivation of RUNX2. In addition, RUNX2 exerts reciprocal inhibition on SOX9 transactivity. In analyses of the mechanism by which SOX9 regulated RUNX2 function, we demonstrated that SOX9 induced a dosedependent degradation of RUNX2. Although RUNX2 is normally degraded by the ubiquitin-proteasome pathway, we found that SOX9-mediated degradation was proteasome-independent but phosphorylation-dependent and required the presence of the RUNX2 Cterminal domain, which contains a nuclear matrix targeting sequence (NMTS). Furthermore, SOX9 was able to decrease the level of ubiquitinated RUNX2 and direct RUNX2 to the lysosome for degradation. SOX9 also preferentially directed b-catenin, an intracellular mediator of canonical Wnt signaling, for lysosomal breakdown. Consequently, the mechanisms by which SOX9 regulates RUNX2 function may underlie broader signaling pathways that can influence osteochondrogenesis and mesenchymal fate. ß

RIC-3 has been identified as a molecule essential for the recruitment of functional nicotinic acetylcholine receptors composed of ␣7, but it exhibits inhibitory effects on ␣4␤2 or ␣3␤4 receptors. In this study, we investigated the role of RIC-3 in the recruitment of 5-hydroxytryptamine type 3A (5-HT 3A ) receptors to the cell surface. Although RIC-3 is not essential for the surface transport of 5-HT 3A receptors, we found that its presence enhances both receptor transport and function in a concentration-dependent manner. RIC-3 is localized to the endoplasmic reticulum, as evidenced by co-localization with the chaperone molecule, binding protein (BiP). RIC-3 is not detected at significant levels on the cell surface when expressed alone or in the presence of 5-HT 3A . RIC-3 and 5-HT 3A show a low level interaction that is transient (<4 h). That RIC-3 can interact with an endoplasmic reticulum-retained 5-HT 3A construct, combined with the transient interaction observed and lack of significant surface-expressed RIC-3, suggests that RIC-3 may play a role in 5-HT 3A receptor folding, assembly, or transport to the cell surface.The most remarkable morphological feature of the brain is not the highly complex, interconnected neuronal pathways but the enormous number (ϳ10 15 ) of potentially distinct synaptic connections involved in information transfer between neurons. Moreover, synapses are not static and passive translators of information but can change their efficiency of synaptic transmission (1). This process is termed "synaptic plasticity" and is thought to lie at the heart of the capacity of the brain for learning and memory. Synaptic plasticity may reside pre-or postsynaptically (or both), modulating neurotransmitter release or neurotransmitter receptor responses, respectively.A fundamental question in neurobiology is how receptor biogenesis is orchestrated. A vast array of receptor-interacting proteins have been identified as participating in ligand-gated ion channel trafficking and localization (1) and have lead to dramatic advances in our knowledge of synaptic plasticity.The 5-HT 3 1 receptors belong to the Cys loop superfamily of ligand-gated ion channels that includes the nicotinic acetylcholine, GABA A , and glycine receptors. The structural relationship (2-5) of the members of this group suggests that their folding and assembly may involve similar posttranslational chaperone-mediated events (6 -8).The forward transport of these receptors requires the appropriate assembly of specific subunits and release from the endoplasmic reticulum (ER) (6,7). Specific assembly signals have been identified in receptors for GABA A (9), glycine (10), and acetylcholine (11). The export of receptors from the ER represents a critical checkpoint for surface expression, with quality control within the lumen of the ER being performed by the resident chaperone proteins (9, 12). In addition, cytoplasmically exposed ER retention signals within the receptors have been identified as elements that control protein export from the ER (13...

In initial work, we developed a 14-day culture protocol under potential GMP, chemically defined conditions to generate chondroprogenitors from human embryonic stem cells (hESCs). The present study was undertaken to investigate the cartilage repair capacity of these cells. The chondrogenic protocol was optimized and validated with gene expression profiling. The protocol was also applied successfully to two lines of induced pluripotent stem cells (iPSCs). Chondrogenic cells derived from hESCs were encapsulated in fibrin gel and implanted in osteochondral defects in the patella groove of nude rats, and cartilage repair was evaluated by histomorphology and immunocytochemistry. Genes associated with chondrogenesis were upregulated during the protocol, and pluripotency-related genes were downregulated. Aggregation of chondrogenic cells was accompanied by high expression of SOX9 and strong staining with Safranin O. Culture with PluriSln1 was lethal for hESCs but was tolerated by hESC chondrogenic cells, and no OCT4-positive cells were detected in hESC chondrogenic cells. iPSCs were also shown to generate chondroprogenitors in this protocol. Repaired tissue in the defect area implanted with hESC-derived chondrogenic cells was stained for collagen II with little collagen I, but negligible collagen II was observed in the fibrin-only controls. Viable human cells were detected in the repair tissue at 12 weeks. The results show that chondrogenic cells derived from hESCs, using a chemically defined culture system, when implanted in focal defects were able to promote cartilage repair. This is a first step in evaluating these cells for clinical application for the treatment of cartilage lesions. STEM CELLS

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RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT 3 receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse "slicks" and bright "halo" structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiledcoil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition.The ligand-gated ion channels are critical participants in cellular communication, playing a major role in synaptic transmission. The ligand-gated ion channels include receptors for acetylcholine (nACh) 3 , ␥-aminobutyric acid type A, serotonin (5-HT), and glycine (the cys-loop superfamily) and N-methyl-D-aspartic acid, kainate, and AMPA (glutamate receptors). A vast array of receptor-interacting proteins have been identified as participating in receptor trafficking and localization (1) and have led to dramatic advances in our knowledge of synaptic architecture and plasticity.Chaperone molecules play an important role in the intracellular transport of receptors. However, a fundamental question in neurobiology remains: How is receptor biogenesis orchestrated? Despite the fact that receptor composition determines receptor function and their pharmacological repertoire, little information is available regarding how this may be achieved. Specific assembly signals exist that can determine a preference for particular subunit partners (2). However, few selective chaperone molecules have been implicated in the process of receptor biogenesis. General chaperone proteins such as BiP, calnexin, calreticulin, and PDI do operate on the ligand-gated ion channels but offer no specificity. In contrast, molecules such as stargazin (3), PSD-95 (4), 14-3-3 (5), and RIC-3 (6) (7-12) are beginning to offer insight into protein-specific ch...

Mesenchymal stem cells (MSCs) are under the control of a large number of signaling systems. In this study, the presence and functionality of the acetylcholine (ACh) signaling system in MSCs was examined. We detected the expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and the presence of ACh in MSCs. MSCs also expressed the nicotinic acetylcholine receptor subunits alpha 3, alpha 5, alpha 7, and the muscarinic acetylcholine receptor 2 (M2-receptor). The M2-receptor and the nicotinic alpha 7 receptor subunits were expressed on distinct subpopulations of cells, indicating differential regulation of cholinergic signaling between MSCs. Stimulation of MSCs with the nicotinic receptor agonist nicotine and the muscarinic receptor agonist muscarine induced immediate and transient increases in intracellular Ca(2+) concentration. Furthermore, muscarine had an inhibiting effect on the production of the intracellular signaling molecule cyclic adenosine 3',5'-monophosphate (cAMP). The AChE inhibitor chlorpyrifos, which is widely used as an agricultural insecticide, had similar effects on intracellular Ca(2+) and cAMP in MSCs. Nicotine, muscarine, and chlorpyrifos induced the phosphorylation of extracellular signal-regulated kinases 1 and 2. This study demonstrates that several components of a cholinergic signaling system are present and functional in MSCs. Environmental compounds such as nicotine and agricultural insecticides can interfere with this system and may affect cellular processes in the MSC.

Human embryonic stem cells (hESCs) have great potential for the repair of damaged articular cartilage. We developed a serum-free 14-day protocol for hESC differentiation into chondrocyte progenitors, which surprisingly lacked strong cartilage matrix production in in vitro tests. In order to direct these progenitors to a more mature phenotype, we investigated substituting different members of the TGFβ family in the protocol. Initially, we supplemented, or substituted GDF5 (day 11–14), with combinations of BMP7 and TGFβ-1, or −3, but these modifications yielded no improvement in matrix gene expression. However, replacing BMP4 with BMP2 (days 3–10 of the protocol) resulted in a more rapid increase in SOX9 gene expression and increased expression of chondrogenic genes SOX5, ACAN and COL2A1. The replacement of BMP4 with BMP2 also enhanced the formation of chondrogenic cell aggregates, with greater deposition of type II collagen. This change was not accompanied by hypertrophic chondrocyte marker COL10A1 expression. The results demonstrate that BMP2 has greater specificity for the generation of chondrogenic cells from hESCs than BMP4 and this was consistent in two hESC lines (HUES1 and MAN7). hESC-chondrogenic cells derived with either BMP2 or BMP4 were tested in vivo by implanting them in fibrin into osteochondral defects in the femur of RNU rats. Repaired cartilage tissue, positive for Safranin O and type II collagen was detected at 6 and 12 weeks with both cell sources, but the BMP2 cells scored higher for tissue quality (Pineda score). Therefore, BMP2 is more effective at driving chondrogenic differentiation from human pluripotent stem cells than BMP4 and the effect on the resulting chondroprogenitors is sustained in an in vivo setting.

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