Effects of Notch ligand Wnt and Sonic Hedgehog Shh Signals on Self Renewal of HSCs

Besides cytokines and molecules consisting of the extracellular matrix, various stimuli such as the Notch ligand, Wnt, and Shh are transmitted to HSCs in the BM microenvironment. The activation of Notch transmembrane receptors expressed on HSCs by their ligand (Delta or Jagged) expressed on stromal cells promotes self-renewal of HSCs [6771]. Karanu et al. reported that a soluble form of Jagged-1 can enhance the expansion of human CD34+ HSCs when added to liquid cultures with SCF, FL, IL-6, IL-3, and G-CSF, indicating the potential usefulness of soluble Jagged-1 for promoting ex vivo expansion of HSCs. It was also reported that a soluble form of Delta-like1 augmented cytokine-dependent ex vivo expansion of HSCs in CB CD133+ cells measured by SRCs [17]. However, since Notch-1 has propensity to induce lymphoid differentiation rather than myeloid differentiation, further studies are required to verify the usefulness and to establish the utility of these approaches. Nonetheless, these strategies would be a promising method to expand HSCs ex vivo. As for the critical target molecule of Notch signals that mediates self-renewal of HSCs, we found that c-Myc was transcriptionally induced by Notch [72]. In addition, the ectopic expression of c-Myc induced the growth of HSCs without disrupting their biologic properties in terms of surface phenotypes, colony-forming abilities, and reconstituting abilities. Thus, c-Myc was supposed to play a major role in self-renewal of HSCs as an effector molecule of Notch signals.

Like Jagged 1/Notch, a number of Wnt proteins are expressed in the BM and their receptor frizzled was detectable on BM-derived HSC/HPCs [73,74]. In the absence of Wnt-mediated signaling, P-catenin is degraded by the ubiquitin/proteosome pathway. Wnt signaling through frizzeled inhibits the degradation of P-catenin, resulting in the accumulation of P-catenin associating with T-cell factor (TCF)/lymphoid-enhancer-binding factor (LEF)-family transcription factors, and these proteins regulate the transcription of downstream target genes. As for the effects of Wnt on HSCs, purified Wnt3a was shown to expand HSCs isolated from Bcl2-transgenic mice ex vivo [75]. In addition to Wnt3a that activates the canonical pathway through Frizzled/p-catenin/TCF/LEF, non-canonical Wnt, Wnt-5a, was also reported to expand HSCs in vitro [76]. However, its mechanisms remain to be clarified. Also, retrovirally expressed a constitutively active form of P-catenin enhanced proliferation of a phenotypically defined murine HSC population [77]. Limiting dilution assays indicated that the induction of activated P-catenin led to over 50-fold increase in HSC numbers after 1-week culture. As for the mechanism of Wnt-mediated proliferation of HSCs, it was demonstrated that the activation of Wnt signaling induced the increased expression of HoxB4 and Notch1 in HSCs,. Glycogen synthase kinase-3(GSK-3) is a constitutively active serine-threonine kinase, which form the destruction complex with Axin and adenomatous polyposis coli (APC). Association with this complex leads to ubiquitilation of P-catenin and subsequent proteosomal degradation. Recently, Trowbridge et al. reported beneficial effects of post transplantation treatment with an ATP-competitive GSK-3 inhibitor on human HSC engraftment [78]. These reports suggested that Wnt signaling is important for the in vitro and in vivo self-renewal of HSCs. However, it is also reported that constitutive activation of P-catenin enforced cell cycle entry of HSCs, thereby, exhausting the long-term repopulating cell pool and leading to hematopoietic failure associated with loss of multilineage differentiation [79,80]. Therefore, fine-tuned (in terms of expression level and duration) Wnt stimulation is required for normal hematopoiesis and critical for therapeutic HSC expansion.

Shh is a family member of human homologs of Drosophila Hedgehog (Hh) and expressed on the cell surface as transmembrane proteins. Hh signals can be mediated through cell-to-cell contact between adjacent cells expressing the Patched (Ptc) receptor. Alternatively, NH2-terminal cleavage of Hh can generate a soluble Hh ligand that can interact with distal cells expressing Ptc [81,82]. In the BM, Shh and their receptors Ptc and Smoothened (Smo) are expressed in highly purified HSCs. Cytokine-induced proliferation of HSCs was inhibited by the anti-Hh Ab, implying that endogenously produced Hh proteins play a role in the expansion of HSCs. Conversely, the addition of soluble forms of Shh increased the number of HSCs with pluripotent repopulating abilities. In addition, Noggin, a potent BMP-4 inhibitor, was found to inhibit the mitogenic effects of Shh, indicating that Shh signaling acts upstream of BMP-4 signaling in the proliferation of HSCs [83].

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