Nov/CCN3 Enhances Long-Term Repopulating Activity of Mouse Hematopoietic Stem Cells Via Intergin β3 Signaling Collaborating with Thrombopoietin

Abstract 862

Hematopoietic stem cells (HSCs) govern hematopoiesis by giving rise to lymphoid, myeloid, and erythroid cells throughout adult life. HSCs reside in a specialized microenvironment termed “niche,” where their functions are regulated by several factors such as cytokines and extracellular matrix (ECM). Nov (CCN3), a member of the CCN family, is a well-known soluble factor that regulates several biological events by binding to integrin receptors, growth factors, and ECM. Recently, Nov has been reported to function as a positive regulator of HSCs, as evidenced by enhanced HSC activity in response to enforced Nov expression in HSCs (Gupta et al., Science, 2007). Furthermore, Nov has been identified as a target gene for Hoxb4, a transcription factor that governs the self-renewal capacity of HSCs (Ohshima et al., Blood, 2011). In addition, Nov expression in HSCs is upregulated by IL-3 via STAT5 activation (Kimura et al., J. Biol. Chem., 2010). Thus, models for the regulation of Nov expression in HSCs have been proposed; however, the mechanisms underlying the regulation of HSC functions by Nov remain unclear. Here, we present a novel mechanism for the enhancement of HSC activity by Nov.

We first suspected that thrombopoietin (TPO), an essential cytokine for HSC maintenance, may promote Nov expression, given that TPO not only induces STAT5 activation in HSCs (Seita et al., PNAS, 2007) but also stimulates Hoxb4 expression (Kirito et al., Blood, 2003). Therefore, we examined the expression of Nov by real-time quantitative RT-PCR in CD150⁺ CD34⁻ c-kit⁺ Sca-1⁺ lineage⁻ (CD150⁺ CD34⁻ KSL) HSCs that were treated with TPO. Similar to IL-3, TPO significantly enhanced Nov expression, compared to that in fresh unstimulated HSCs (p < 0.05). In contrast, stem cell factor (SCF), a critical cytokine for the maintenance of HSC functions, completely lost Nov expression. This strong link between Nov and TPO in HSCs suggests that TPO may play a key role in the regulation of HSCs by Nov. Therefore, we examined the long-term repopulating (LTR) activity of HSCs in transplantation assays following treatment with exogenous Nov in the presence of TPO or SCF. Interestingly, TPO stimulation supported the Nov-induced enhancement of HSC LTR activity (p < 0.05), whereas this positive effect was completely abolished in the presence of SCF. Furthermore, treatment with TPO, but not with SCF, increased the capture of Nov by HSCs, as measured by flow cytometry analyses using Alexa647-labeled Nov (p < 0.001), which strongly suggests that the positive effect of exogenous Nov on the LTR activity of HSCs is specifically dependent on TPO. More importantly, this TPO-mediated promotion of Nov binding to HSCs was blocked by antibodies against integrin αv or β3, indicating that integrin avβ3 is the primary receptor for Nov on HSCs.

Previously, we demonstrated that outside-in signaling via phosphorylated Tyr747 of integrin 3 (β3^PY747) is indispensable for the TPO-dependent maintenance of mouse HSCs, which requires the activation (conformational change for high affinity ligand binding) of αvβ3 integrin via TPO-induced inside-out signaling (Umemoto et al., Blood ASH abstract, 2009). Given our previous data, the results from the present study suggest that Nov regulates the LTR activity of HSCs through outside-in signaling especially via β3^PY747, following its ligation to integrin αvβ3 that has been activated by TPO-induced inside-out signaling.

Finally, we confirmed our hypothesis by using β3 integrin mutant mice that harbor an alanine substitution of tyrosine 747 in the cytoplasmic tail of β3 integrin (Y747A), which impairs integrin inside-out and outside-in signaling. Transplantation assays using Y747A-expressing HSCs revealed that inhibition of bidirectional integrin signaling by the Y747A mutation completely abolished the positive TPO-dependent effect of Nov, even when αvβ3 integrin activation was rescued by Mn²⁺, an external inducer of integrin activation that acts independently of inside-out signaling.

Taken together, our findings demonstrate that Nov positively regulates HSC activity through outside-in signaling via β3^PY747, following its TPO-dependent ligation to integrin αvβ3. Thus, we present a novel mechanistic link between Nov, β3 integrin, and TPO in HSCs.