Elsevier

Cellular Signalling

Volume 23, Issue 8, August 2011, Pages 1358-1365
Cellular Signalling

Rb and nucleolin antagonize in controlling human CD34 gene expression

https://doi.org/10.1016/j.cellsig.2011.03.018Get rights and content

Abstract

Retinoblastoma protein (Rb) controls cell proliferation, differentiation, survival and gene expression and it has a central role in the signaling network that provides a cell cycle checkpoint in the G1 phase of the cell cycle. Studies in mice have shown that Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment and it acts as a critical regulator of hematopoietic stem and progenitor cells under stress. In human hematopoiesis, the CD34 protein is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation, and hematopoietic reconstitution, and CD34 has a role in the differentiation of hematopoietic cells. Here we find that, in CD34-positive hematopoietic cells, Rb controls the human CD34 promoter region by antagonizing the CD34 promoter factor nucleolin to provide a mechanism that links expression of endogenous CD34 to cell cycle progression. Our study suggests a direct involvement of Rb in the transcriptional program of human CD34-positive hematopoietic stem/progenitor cells, thus providing further insights into the molecular network relevant to the features of these cells.

Introduction

Rb controls cell proliferation, differentiation, survival and gene expression and it has a central role in the signaling network that provides a cell cycle checkpoint in the G1 phase of the cell cycle [1], [2], [3], [4], [5]. In its hypophosphorylated form, Rb restrains cell proliferation, in part by targeting the E2F transcription factors, and phosphorylation of Rb by cyclin-dependent kinases (Cdks) is associated with G1/S transition and S phase progression [4], [5], [6], [7], [8]. Rb is a prototypical tumor suppressor and a member of a family of related proteins that also includes the p107 and p130 protein [1], [3], [4], [5], [9]. Due to the significance of the signaling network associated with Rb to cell-fate decisions, it is important to work out Rb functions in stem and progenitor cells to better understand both tissue homeostasis and cancer disease process [4], [10], [11]. Recent studies investigated the role of Rb in hematopoietic stem and progenitor cells using mouse models [12], [13], [14], [15]. It has been shown that Rb regulates the interactions between hematopoietic stem cells (HSCs) and the bone marrow (BM) microenvironment and it acts as a critical regulator of hematopoietic stem and progenitor cells in response to stress signals [13], [14]. Simultaneous inactivation of all Rb family proteins in mouse HSCs leads to a loss of HSC quiescence, hyperproliferation of early hematopoietic progenitors and to imbalance between lymphoid and myeloid cell fates [15]. Quiescent mouse HSCs can be reversibly activated to switch from dormancy to self-renewal in response to injury cues, and a strong upregulation of the CD34 gene transcripts is observed in activated HSCs [16].

In human hematopoiesis, CD34 glycoprotein is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation and reconstitution of the hematopoietic system [17]. Experimental evidence for involvement of CD34 in hematopoietic reconstitution and differentiation of hematopoietic cells have been presented [18], [19]. The expression of cell surface CD34 glycoprotein is broadly utilized for enumeration of stem/progenitor cells for clinical BM transplantation, and the number of 5-year survivors of HSC transplantation is estimated to be 100,000 worldwide [20]. In this context, it is important to figure out mechanisms that control CD34 gene expression to understand the processes underlying the complex functions of hematopoietic stem/progenitor cells at a molecular level.

Regulation of the human CD34 gene has been investigated as a model of hematopoietic stem/progenitor cell-specific gene control, and transcriptional as well as post-transcriptional mechanisms have been identified [17], [21], [22], [23], [24], [25], [26], [27], [28]. It has been shown that multiple regulatory elements are necessary to provide proper transcriptional control of the CD34 gene [22], [23], [24]. Furthermore, transcription regulators including c-Myb, Ets-2, MZF-1 and nuclear factor Y have been shown to bind to their cognate sites and modulate human CD34 promoter region activity [25], [26], [27], [28]. In addition, nucleolin is recruited to the CD34 promoter region in human CD34-positive hematopoietic cells to activate CD34 gene transcription [29]. Nucleolin is a multifunctional protein, which is found in the nucleolus, in the nucleoplasm and in the cytoplasm, and it is abundant in growing and cancer cells [30], [31], [32]. The involvement of nucleolin in regulation of gene transcription, chromatin remodeling and RNA metabolism has been described [32], [33], [34], [35], [36], [37]. Our recent study reported a cell cycle-controlled interaction of nucleolin with Rb in epithelial cells whereby Rb inhibits transcription of the human papillomavirus type 18 (HPV18) oncogenes [38]. In hematopoietic tissue, human nucleolin is enriched in CD34-positive cells, as opposed to CD34-negative mononuclear cells [29]. Nucleolin gene transcripts are also selectively enriched in murine BM HSCs, as opposed to differentiated hematopoietic cells, and nucleolin protein is present at higher levels in murine long-term reconstituting HSCs versus non-long-term reconstituting progenitor cells [39], [40].

Here we find that, in CD34-positive hematopoietic cells, Rb controls the human CD34 promoter region by antagonizing the CD34 promoter factor nucleolin to provide a mechanism that links expression of endogenous CD34 to cell cycle progression. Our study suggests a direct involvement of Rb in the transcriptional program of human CD34-positive hematopoietic stem/progenitor cells, thus providing further insights into the molecular network relevant to the features of these cells.

Section snippets

Cell culture and flow cytometry

Human BM-derived CD34-positive myeloblast cell line KG1 [41] was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. KG1 cells stably transfected with the pCMV-Tag2B expression vector containing the full-length human nucleolin cDNA were described [29]. Detailed cell synchronization conditions and the necessary controls were described [42], [43]. In brief, G1 phase cells were prepared by 20 μM lovastatin treatment for 24 to 36 h, and S phase cells were prepared by 3 μM

Rb controls human CD34 promoter region by antagonizing nucleolin activity

To investigate a possible involvement of Rb in the control of human CD34 gene promoter region, co-transfection experiments were performed on the use of a CD34 promoter reporter construct and the human BM-derived CD34-positive myeloblast cell line KG1 [41]. The CD34 promoter reporter construct comprised nucleotide numbers − 666 to + 175 of the CD34 gene promoter region [22], referred to as CD34wt-pGL3. The experiments employed either an expression vector encoding the full-length WT Rb cDNA or an

Discussion

Here we find that Rb controls the human CD34 promoter region by antagonizing the CD34 promoter factor nucleolin. Our recent study reported that nucleolin can be viewed as a component of the gene regulation program of CD34-positive hematopoietic cells, and it is recruited to the CD34 promoter in these cells [29]. It has been described that, via thus far incompletely defined mechanisms, the expression of CD34 by stem/progenitor cells comes along with induction of genes related to self-renewal,

Conclusions

In summary, the present study reveals that, in CD34-positive hematopoietic cells, Rb controls the human CD34 promoter region by antagonizing the CD34 promoter factor nucleolin to provide a mechanism that links expression of endogenous CD34 to cell cycle progression. Our study suggests a direct involvement of Rb in the transcriptional program of human CD34-positive hematopoietic stem/progenitor cells, thus providing further insights into the molecular network relevant to the features of these

Acknowledgements

We thank Prof. Kristian Helin for Rb expression constructs, Prof. Daniel G. Tenen for CD34 promoter construct and Prof. Elmar Gren for helpful discussions.

This work was funded by the Deutsche Forschungsgemeinschaft (DFG) grant GR 3581/2-1 to E. Grinstein.

A. Borkhardt was supported by the German-Israeli Foundation (GIF).

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