Yuhan Kong

Wnt signaling in bone formation and its therapeutic potential for bone diseases

The Wnt signaling pathway plays an important role not only in embryonic development but also in the maintenance and differentiation of the stem cells in adulthood. In particular, Wnt signaling has been shown as an important regulatory pathway in the osteogenic differentiation of mesenchymal stem cells. Induction of the Wnt signaling pathway promotes bone formation while inactivation of the pathway leads to osteopenic states. Our current understanding of Wnt signaling in osteogenesis elucidates the molecular mechanisms of classic osteogenic pathologies. Activating and inactivating aberrations of the canonical Wnt signaling pathway in osteogenesis results in sclerosteosis and osteoporosis respectively. Recent studies have sought to target the Wnt signaling pathway to treat osteogenic disorders. Potential therapeutic approaches attempt to stimulate the Wnt signaling pathway by upregulating the intracellular mediators of the Wnt signaling cascade and inhibiting the endogenous antagonists of the pathway. Antibodies against endogenous antagonists, such as sclerostin and dickkopf-1, have demonstrated promising results in promoting bone formation and fracture healing. Lithium, an inhibitor of glycogen synthase kinase 3β, has also been reported to stimulate osteogenesis by stabilizing β catenin. Although manipulating the Wnt signaling pathway has abundant therapeutic potential, it requires cautious approach due to risks of tumorigenesis. The present review discusses the role of the Wnt signaling pathway in osteogenesis and examines its targeted therapeutic potential.

Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells.

Growth hormone (GH) is usually released by somatotrophs in the anterior pituitary in response to the GH‐releasing hormone and plays an important role in skeleton development and postnatal growth. However, it is unclear if extrapituitary GH exerts any effect on murine multilineage cells (MMCs). MMCs are multipotent progenitors that give rise to several lineages, including bone, cartilage, and fat. We have identified bone morphogenic protein 9 (BMP9) as one of the most osteogenic BMPs in MMCs by regulating a distinct set of downstream mediators. In this study, we find that GH is one of the most significantly upregulated genes by BMP9 in mouse MMCs through expression‐profiling analysis. We confirm that GH is a direct early target of and upregulated by BMP9 signaling. Exogenous GH synergizes with BMP9 on inducing early and late osteogenic markers in MMCs. Furthermore, BMP9 and GH costimulation leads to a significant expansion of growth plate in cultured limb explants. Although GH alone does not induce de novo bone formation in an ectopic bone formation model, BMP9 and GH costimulated MMCs form more mature bone, which can be inhibited by silencing GH expression. The synergistic osteogenic activity between BMP9 and GH can be significantly blunted by JAK/STAT inhibitors, leading to a decrease in GH‐regulated insulin‐like growth factor 1 (IGF1) expression in MMCs. Our results strongly suggest that BMP9 may effectively regulate extrapituitary GH expression in MMCs. Thus, it is conceivable that the BMP9‐GH‐IGF axis may be exploited as an innovative strategy to enhance osteogenesis in regenerative medicine.

BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells

Summary Mesenchymal stromal progenitor cells (MSCs) are multipotent progenitors that can be isolated from numerous tissues. MSCs can undergo osteogenic differentiation under proper stimuli. We have recently demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most osteogenic BMPs. As one of the least studied BMPs, BMP9 has been shown to regulate angiogenesis in endothelial cells. However, it is unclear whether BMP9-regulated angiogenic signaling plays any important role in the BMP9-initiated osteogenic pathway in MSCs. Here, we investigate the functional role of hypoxia-inducible factor 1&agr; (HIF1&agr;)-mediated angiogenic signaling in BMP9-regulated osteogenic differentiation of MSCs. We find that BMP9 induces HIF1&agr; expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1&agr; potentiates BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo. siRNA-mediated silencing of HIF1&agr; or HIF1&agr; inhibitor CAY10585 profoundly blunts BMP9-induced osteogenic signaling in MSCs. HIF1&agr; expression regulated by cobalt-induced hypoxia also recapitulates the synergistic effect between HIF1&agr; and BMP9 in osteogenic differentiation. Mechanistically, HIF1&agr; is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling and osteogenic signaling in MSCs. Thus, our findings should not only expand our understanding of the molecular basis behind BMP9-regulated osteoblastic lineage-specific differentiation, but also provide an opportunity to harness the BMP9-induced synergy between osteogenic and angiogenic signaling pathways in regenerative medicine.

Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9‐induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9‐induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre‐osteoblast C2C12 cells. BMP9‐induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7‐induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9‐induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2‐induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9‐induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9s potent osteogenic capability in MSCs.

Cross‐talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross‐talks with BMP9 and regulates BMP9‐induced osteogenic differentiation. We find that EGF potentiates BMP9‐induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG‐1478 and AG‐494 in a dose‐ and time‐dependent manner. Furthermore, EGF significantly augments BMP9‐induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9‐induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up‐regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross‐talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.

Conditional Immortalization Establishes a Repertoire of Mouse Melanocyte Progenitors with Distinct Melanogenic Differentiation Potential

TO THE EDITOR Epidermal melanocytes (MCs) are specialized melanin-producing cells (Slominski et al., 2004; Yamaguchi et al., 2007; Thomas and Erickson, 2008; Park et al., 2009) that synthesize melanin within the melanosome (Slominski et al., 2004) and protect individuals from harmful UV rays (Slominski et al., 2004; Yamaguchi et al., 2007; Thomas and Erickson, 2008; Park et al., 2009). Defects in or a lack of MCs can lead to melanoma, pigment disorders, and auditory defects. MC proliferation and differentiation in skin is tightly linked to hair regeneration cycles. MCs in vertebrates are derived from neural crest (Thomas and Erickson, 2008). In hair follicles, melanoblasts are segregated into hair matrix MCs (for hair pigmentation) and melanocyte stem cells (MCSCs). The discovery of MCSCs and induced pluripotent (iPS) cells provides important resources to elucidate mechanisms underlying melanogenesis and pathogenesis of MC-related disorders (Lin and Chuong, 2011; Nishikawa-Torikai et al., 2011; Nishimura, 2011; Ohta et al., 2011; Yang et al., 2011). Although MCSCs and iPS cells are important MC precursors for mechanistic studies, their isolation and expansion are technically challenging. Here we investigate whether MCs can be immortalized without compromising melanogenic potential. By using primary MCs isolated from newborn mouse skin, we engineered >100 clones by introducing SV40 T antigen (SV40T), which is flanked with FRT sites (Supplementary Figure S1A online; Westerman and Leboulch, 1996). We found that although primary MCs grew slower after passage 5 immortalized MC (iMC) cells acquired high proliferative activity (Supplementary Figure S1B online); however, wide variations in proliferation were observed among clones. For instance, iMC23 grew faster than iMC65 (Figure 1a). Figure 1 Characterization of SV40T-immortalized melanocytes (iMCs) We analyzed melanogenic markers in iMC clones. More than half of the analyzed clones expressed progenitor marker c-kit, whereas MC progenitor markers Pax3, Sox10, and MITF-m were readily detected in most clones (Supplementary Figure S2A online). Dopachrome tautomerase and tyrosinase-related protein 1 (TRP-1) were highly expressed in most iMC clones, whereas tyrosinase was highly expressed in about half of the clones (Supplementary Figure S2B and C online). It is conceivable that early melanogenic iMCs should express c-kit and early melagonic markers but not late markers (e.g., tyrosinase). Three iMC clones were chosen for further characterization: iMC23 (melanoblast progenitor-like) (Figure 1aA), iMC65 (late-stage melanocyte-like) (Figure 1aB), and iMC37 (intermediately differentiated melanoblast-like). These results indicate that SV40T-mediated immortalization can create a repertoire of iMCs with varied melanogenic potential. We also tested whether immortalization phenotypes were reversible by flippase recombination enzyme (FLP). By using adenovirus FLP (AdFLP) that co-expresses green fluorescent protein (GFP; He et al., 1998; Luo et al., 2007), we found that iMCs were effectively transduced by AdFLP or AdGFP, and SV40T expression was reduced in FLP-transduced iMCs (Figure 1bA and bB). iMC23 infected with AdFLP grew slower (Figure 1bC and bD), suggesting that the proliferative activity of iMCs may be reversed by removing SV40T. By using reporter pTyr-Gluc-expressing Gaussia luciferase (GLuc) driven by a 2.0-kb mouse tyrosinase promoter, we found that GLuc activity was increased by dexamethasone and was potentiated by removing SV40T with FLP (Supplementary Figure S3A and B online). We further analyzed spontaneous differentiation of iMCs by assessing endogenous melanin and tyrosinase activity. Cell pellets from iMCs exhibited varied amounts of melanin. iMC65 and iMC61 exhibited the highest level of melanin and iMC23 produced the lowest level, whereas a majority of iMC clones produced low-to-modest levels of melanin (Figure 1cA). Accordingly, quantitative analysis revealed that iMC65 and iMC61 exhibited high tyrosinase activity, and seven clones including iMC23 had low tyrosinase activity, whereas iMC37 exhibited modest-to-high levels of tyrosinase activity (Figure 1cB), consistent with the qualitative results shown in Figure 1cA. We characterized the early and late markers c-kit and HMB45, respectively, in iMC23, iMC37, and iMC65. HMB45 is a widely used mAb detecting melanocytic tumors (Gown et al., 1986). iMC23 expressed a high level of c-kit, whereas iMC37 and iMC65 were strongly stained with HMB45 (Figure 1dA). Fontana–Masson staining revealed the highest level of melanin in iMC65 and a modest level in iMC37, whereas a negligent level of melanin was observed in iMC32 (Figure 1dB). Thus, these clones may represent different stages of melanogenic differentiation. We further analyzed the melanogenic potential of iMC23. When iMC23 was treated with dexamethasone, tyrosinase activity increased in a dose-dependent manner (Figure 2aA). Dexamethasone induced tyrosinase and TRP-1 expression (Figure 2aB). Dose-dependent melanin production was visible in culture and cell pellet (Figure 2bA and bB), which was further confirmed by Fontana–Masson staining (Figure 2bC). These results indicate that iMC23 exhibits melanogenic potential. Figure 2 Melanogenic potential of immortalized melanocytes (iMCs) Finally, we determined whether iMC immortalization phenotypes were reversible in vivo. iMC23 and iMC65 tagged with firefly luciferase were infected with AdFLP or AdGFP, collected for subcutaneous injection into athymic mice, and monitored by Xenogen bioluminescence imaging (Caliper Life Sciences, Hopkinton, MA). AdGFP-transduced iMC23 yielded a stronger signal than that of AdFLP-transduced iMC65, whereas within the same lines AdFLP-transduced cells produced no signal compared with the AdGF-transduced iMC65 (Figure 2cA), suggesting that removing SV40T may reduce proliferation and survival of iMCs in vivo. Pigmentation of injected iMCs was visible through skin, as iMC65 exhibited a higher level of pigmentation than iMC23. AdFLP-transduced iMC23 yielded more pigmentation than AdGFP-transduced iMC23 (Figure 2cB). Histologically, iMC23 injection sites had higher cellularity than iMC65 sites, whereas in both lines cellularity was lower in AdFLP-transduced cells (Supplementary Figure S3C online). Production of melanin was more pronounced in AdFLP-transduced iMCs as indicated by HMB45 immunostaining and Fontana–-Masson staining (Figure 2dA and dB). Overall, the in vivo results are consistent with these clones’ in vitro features. SV40T-immortalized MCs are non-tumorigenic under our experimental conditions. Interestingly, melanoma phenotypes have been reported by enabling neoplastic transformation of primary human MCs with SV40 early region, which encodes both SV40 T and t antigens, in conjunction with hTERT (Gupta et al., 2005). Nevertheless, we established a repertoire of conditionally immortalized MCs with varied melanogenic potential, ranging from melanoblast-like to well-differentiated phenotypes. Such repertoire of iMCs should be useful for understanding MC biology and raveling molecular pathogenesis of pigment cell disorders, including melanoma. An efficient method to isolate and expand cutaneous MCs is needed, as it enables us to better understand melanogenesis. Here we demonstrate that SV40T-mediated immortalization of MCs is simplistic, effective, and reversible. This approach should be considered an important alternative to the isolation and characterization of melanogenic stem cells.

Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein

Background and Aims: Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3βmay play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins. Methods and Results: We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion: Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling.

Endoplasmic Reticulum (ER) Stress Inducible Factor Cysteine-Rich with EGF-Like Domains 2 (Creld2) Is an Important Mediator of BMP9-Regulated Osteogenic Differentiation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.