Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

Wnts were previously shown to regulate the neurogenesis of neural stem or progenitor cells. Here, we explored the underlying molecular mechanisms through which Wnt signaling regulates neurotrophins (NTs) in the NT-induced neuronal differentiation of human mesenchymal stem cells (hMSCs). NTs can increase the expression of Wnt1 and Wnt7a in hMSCs. However, only Wnt7a enables the expression of synapsin-1, a synaptic marker in mature neurons, to be induced and triggers the formation of cholinergic and dopaminergic neurons. Human recombinant (hr)Wnt7a and general neuron makers were positively correlated in a dose- and time-dependent manner. In addition, the expression of synaptic markers and neurites was induced by Wnt7a and lithium, a glycogen synthase kinase-3β inhibitor, in the NT-induced hMSCs via the canonical/β-catenin pathway, but was inhibited by Wnt inhibitors and frizzled-5 (Frz5) blocking antibodies. In addition, hrWnt7a triggered the formation of cholinergic and dopaminergic neurons via the non-canonical/c-jun N-terminal kinase (JNK) pathway, and the formation of these neurons was inhibited by a JNK inhibitor and Frz9 blocking antibodies. In conclusion, hrWnt7a enhances the synthesis of synapse and facilitates neuronal differentiation in hMSCS through various Frz receptors. These mechanisms may be employed widely in the transdifferentiation of other adult stem cells

Sexually transmitted disease surveillance 2007

Division of STD Prevention."December 2008."Also available via the World Wide Web as an html or an Acrobat .pdf file (7.3 MB, 194 p.).Centers for Disease Control and Prevention. Sexually Transmitted Disease Surveillance, 2007. At lanta, GA: U.S. Department of Health and Human Services; December 2008

Signal peptide-CUB-EGF-like repeat-containing protein 1-promoted FLT3 signaling is critical for the initiation and maintenance of MLL-rearranged acute leukemia

A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia that offers an opportunity for targeted therapy is addiction to protein tyrosine kinase signaling. One such signal is the receptor tyrosine kinase Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family proteins have previously been shown to act as a co-receptor for augmenting signaling activity of a receptor tyrosine kinase (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo acute myeloid leukemia. We further demonstrate, by using a conditional knockout mouse model, that Scube1 is required for both the initiation and maintenance of MLL-AF9-induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses revealed that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a non-receptor tyrosine kinase) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody-drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study indicates a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r acute myeloid leukemia. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements

Mechanostimulation-induced integrin αvβ6 and latency associated peptide coupling activates TGF-β and regulates cancer metastasis and stemness

The existence of cancer stem cells is the single most important factor contributing to cancer recurrence, and despite immense therapeutic relevance, little research has been done on investigating their origin. Through mechanotransduction, cells translate biophysical cues to biochemical signals. However, little is known about its role in acquisition of cancer stem cell characteristics in non-stem cells. Here, highly ordered nanoenvironments are engineered as models to induce mechanotransduction in cancer cells and elucidate how cell environment delivers precise physical cues via mechanotransduction to modulate expression and localization of key mesenchymal markers to induce epithelial-mesenchymal transition (EMT) and regulate cancer stemness. By initiating integrin αVβ6 and Latency associated peptide (LAP) interactions, cell nanoenvironment mechanically activates TGF-β canonical and non-canonical signaling pathways and induces Epithelial-Mesenchymal transition in U2OS osteosarcoma cells. As a consequence of TGF-β mechanical activation, a synchronous regulation in cancer stem-cell and pluripotency biomarkers is also observed which transcends to formation of cell organoids, a characteristic of cancer stem cells. Furthermore, nanoenvironment-derived cells promote tumor growth and metastasis in-vivo. Mechanistically, RNA-sequencing, RNA-interference and protein translocation experiments establish that cell nanoenvironment plays a decisive role in imparting stemness abilities to incoming cells via EMT and reveals how cells can exploit mechanical sensing to orchestrate tumorigenicity

Neurogenic effects from different Wnts in NT-induced hMSCs.

<p>(A) mRNA levels of <i>MAP2</i>, <i>SYN1</i>, and <i>LEF1</i> were quantified after 48 h of different Wnt treatments (2 µg/ml) in hMSCs that had been treated with NTs for 1 week. All Wnts promoted <i>MAP2</i> expression, and Wnt7a induced the highest <i>SYN1</i> expression. Levels were normalized to those of NTs treatments (set to 1.0). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. ¹<i>p</i><0.05, ^1″<i>p</i><0.01 (DMEM vs. all groups); ²<i>p</i><0.05, ^2″<i>p</i><0.01 (NTs vs. all groups); ³<i>p</i><0.05, ^3″<i>p</i><0.01 (Wnt1 vs. all groups); ⁴<i>p</i><0.05, ^4″<i>p</i><0.01 (Wnt3a vs. all groups); ⁵<i>p</i><0.05, ^5″<i>p</i><0.01 (Wnt5a vs. all groups); ⁶<i>p</i><0.05, ^6″<i>p</i><0.01 (Wnt7a vs. all groups). (B) mRNA levels of <i>ChAT</i>, <i>DBH</i>, and <i>LEF1</i> were quantified after 48 h of different Wnt treatments (2 µg/ml) in hMSCs that had been treated with NTs for 1 week. Wnt1 had no effects on <i>ChAT</i> or <i>DBH</i> expressions, but Wnt7a significantly induced both genes. Levels were normalized to those of NTs treatments (set to 1.0). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. ¹<i>p</i><0.05, ^1″<i>p</i><0.01 (DMEM vs. all groups); ²<i>p</i><0.05, ^2″<i>p</i><0.01 (NTs vs. all groups); ³<i>p</i><0.05, ^3″<i>p</i><0.01 (Wnt1 vs. all groups); ⁴<i>p</i><0.05, ^4″<i>p</i><0.01 (Wnt3a vs. all groups); ⁵<i>p</i><0.05, ^5″<i>p</i><0.01 (Wnt5a vs. all groups); ⁶<i>p</i><0.05, ^6″<i>p</i><0.01 (Wnt7a vs. all groups).</p

Inhibitory effects of Wnt inhibitors and blocking antibodies in Wnt7a-induced synapsin expression.

<p>(A) As described in "<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104937#s2" target="_blank">Materials and Methods</a>", mRNA levels of <i>SYN1</i> and <i>LEF1</i> were examined by a qPCR. sFRP4 showed significant inhibition of <i>SYN1</i> expression, and Frz5 blocking antibodies greatly inhibited gene expressions. Levels were normalized to those in NTs groups (set to 1.0). * <i>p</i><0.05, ** <i>p</i><0.01 (NTs+Wnt7a vs. all groups). (B) Percentages of inhibition calculated from (A). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments.</p

Neuronal specification by the non-canonical Wnt7a pathway in NT-induced hMSCs.

<p>(A) mRNA levels of <i>ChAT</i> and <i>DBH</i> were examined in NT-induced hMSCs, and SP600125 (15 µM) and Wnt7a (2 µg/ml) or LiCl (4 mM) were added to NT-induced hMSCs at the same time. Levels were normalized to those in the NTs control (set to 1.0). Wnt7a, but not lithium, stimulated mRNA levels in NT-induced hMSCs, and SP600125 totally inhibited Wnt7a-induced <i>ChAT</i> and <i>DBH</i> expressions. Data are presented as the mean ± SD of one triplicate experiment that was representative of the three independent experiments. * <i>p</i><0.05, ** <i>p</i><0.01 (all vs. NTs). ^#<i>p</i><0.05, ^##<i>p</i><0.01 (all vs. NTs+Wnt7a+SP600125). ⁺<i>p</i><0.05, ⁺⁺<i>p</i><0.01 (all vs. NTs+LiCl+SP600125). (B) p4 NT-treated hMSCs were immunoblotted with ChAT, DBH, and GAPDH. The NT groups were treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for 7 days first, and then with NTs+Wnt7a or lithium for 7 days. DMEM groups served as controls. (C) Expression levels of <i>MAP2</i> and <i>SYN1</i> in NT-induced hMSCs with SP600125/Wnt7a or SP600125/LiCl are shown. SP600125 had no effect in <i>MAP2</i> or <i>SYN1</i> expression. Levels were normalized to those in NTs groups (set to 1.0). * <i>p</i><0.05, ** <i>p</i><0.01 (NTs vs. all groups). ^#<i>p</i><0.05, ^##<i>p</i><0.01 (all vs. NTs+Wnt7a+SP600125). ⁺<i>p</i><0.05, ⁺⁺<i>p</i><0.01 (all vs. NTs+LiCl+SP600125). (D) p4 NT-treated hMSCs were stained with ChAT (green) and DBH (red). NTs groups were treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for the first 7 days and then with NTs+Wnt7a or lithium for the next 7 days. In inhibitory groups, SP600125 was added with Wnt7a or lithium in NT-induced hMSCs at the same time. DAPI (blue) was used as a counterstain. DMEM groups were used as controls. The white bar represents 50 µm. (E) Percentages of ChAT-positive cells and DBH-positive cells among all DAPI-positive cells calculated from (D). All data are presented as the mean ± SD. * <i>p</i><0.05, ** <i>p</i><0.01 (all vs. NTs). ^#<i>p</i><0.05, ^##<i>p</i><0.01 (all vs. NTs+Wnt7a+SP600125). ⁺<i>p</i><0.05, ⁺⁺<i>p</i><0.01 (all vs. NTs+LiCl+SP600125). (F) As described in "<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104937#s2" target="_blank">Materials and Methods</a>", mRNA levels of <i>ChAT</i> and <i>DBH</i> were examined by a qPCR. Levels were normalized to those in NTs groups (set to 1.0). * <i>p</i><0.05, ** <i>p</i><0.01 (NTs+Wnt7a vs. all groups). (G) Percentages of inhibition calculated from (E). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments.</p

Immunostaining and immunoblotting of NT-stimulated hMSCs with Wnt7a or lithium.

<p>(A) p4 NT-treated hMSCs were stained with β-catenin (green). The NTs+Wnt7a groups were treated with NTs+Wnt7a for 24 h, and the NTs+lithium groups were treated with NTs+lithium for 24 h. 4,6-Diamidino-2-phenylindole (DAPI) (blue) was used as a counterstain. (B) p4 NT-treated hMSCs were immunoblotted with MAP2, SYN1, and GAPDH. The NT group was treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for 7 days first, and then with NTs+Wnt7a or lithium for 7 days. DMEM groups served as controls. (C) p4 NT-treated hMSCs were stained with MAP2 (red), SYN1 (red), and β-catenin (green). NTs groups were treated with NTs for 14 days. The NTs+Wnt7a and NTs+lithium groups were treated with NTs for 7 days first, and then NTs+Wnt7a or lithium for 7 days. DAPI (blue) was used as a counterstain. DMEM groups served as the control. The white bar represents 50 µm. (D) Percentages of MAP2-positive cells, SYN1-positive cells, and neurite-positive cells among all DAPI-positive cells. All data are presented as the mean ± SD. * <i>p</i><0.05, ** <i>p</i><0.01 (all vs. NTs). (E) Cell areas were calculated by β-catenin-positive cells from (B).</p

Flow cytometric analysis and Wnt profiles of hMSCs by the induction of NTs.

<p>(A) Bone marrow-derived hMSCs were analyzed following four cell passages. hMSCs were positive for CD44, CD73, CD105, CD166, and Stro-1, and negative for CD14 and CD34. The solid curves indicate each type of antibody, and the filled curves indicate mouse IgG as the negative control. (B) mRNA levels of <i>MAP2</i> were quantified on days 7, 14, and 21 during stimulation with NTs. NTs significantly increased <i>MAP2</i> levels on days 14 and 21. Untreated hMSCs served as the control. (C) mRNA levels of <i>Wnt1</i>, <i>Wnt3a</i>, <i>Wnt5a</i>, <i>Wnt7a</i>, and <i>Wnt7b</i> were quantified on days 7, 14, and 21 during stimulation with NTs. NTs increased the expression of <i>Wnt1</i> and induced expressions of <i>Wnt7a</i> and <i>Wnt7b</i>. * <i>p</i><0.05, ** <i>p</i><0.01 (i.e., treated vs. control in the Wnt1, Wnt3a, and Wnt5a groups; NTs at 14 and 21 days vs. NTs at 7 days in the Wnt7a and Wnt7b groups). Data are presented as the mean ± SD of one triplicate experiment that was representative of three independent experiments. * <i>p</i><0.05, ** <i>p</i><0.01 (i.e., treated vs. control). ND, not determined.</p