Here, we show that the tumor-homing F3 peptide, which binds to and is internalized by endothelial and tumor cells (Porkka et al., 2002
), interacts with nucleolin. We also show that antinucleolin antibodies detect nucleolin at the surface of cultured tumor cells and endothelial cells of angiogenic vessels in vivo. These results support the previously proposed role for nucleolin as a shuttle molecule between the nucleus and the cell surface, and they define cell surface nucleolin as a novel vascular marker for angiogenic endothelium.
Several approaches were used to identify the binding molecule for the F3 peptide as nucleolin. First, nucleolin and histones were identified as the main cellular proteins that specifically bound to immobilized F3 peptide. Cell surface labeling indicated that the bound nucleolin was derived from the surface of intact cells, whereas the histones were not labeled and, therefore, likely originated from dead cells. Second, inhibition of F3 uptake into cultured cells by an antinucleolin antibody that is internalized into the nucleus provides additional evidence for the specificity of the F3–nucleolin interaction and its occurrence in intact cells. Third, the specific binding of injected antinucleolin antibodies to tumor blood vessels extends the association of F3 binding and cell surface nucleolin expression to an in vivo animal model.
The nucleolin polypeptide consists of a negatively charged NH2
-terminal domain, an RNA-binding domain, and a COOH-terminal domain rich in RGG motifs. The main functions of nucleolin relate to rRNA maturation and ribosome assembly (Ginisty et al., 1999
; Srivastava and Pollard, 1999
). Although nucleolin was originally described as a nuclear and cytoplasmic protein, a number of studies show that it can also be expressed at the cell surface (Deng et al., 1996
; Larrucea et al., 1998
; Said et al., 2002
; Sinclair and O'Brien, 2002
). Recent results also ascribe additional functions to nucleolin as a shuttle protein between the cytoplasm and the nucleus (Borer et al., 1989
; Yu et al., 1998
), and between the cell surface and the nucleus (Schmidt-Zachmann and Nigg, 1993
; Said et al., 2002
; Shibata et al., 2002
). The localization of nucleolin within the cell may be regulated by phosphorylation of its NH2
terminus (Schwab and Dreyer, 1997
). Our results provide additional evidence for the cell surface localization and shuttle function of nucleolin.
The expression of nucleolin at the cell surface seems to correlate with growth and metabolic activity of cells. Both the uptake of the F3 peptide and the staining of intact cells with antinucleolin antibodies were suppressed in serum-starved cells. This may be a proliferation-related effect. An association of cell surface nucleolin expression with cell proliferation in vitro has been described previously (Hovanessian et al., 2000
). Other factors besides proliferation may contribute to the regulation of cell surface nucleolin expression. We found only modest levels of cell surface nucleolin on actively proliferating endothelial cells in vitro, whereas antinucleolin binding to angiogenic endothelium was readily detectable in vivo. The differentiation state of the cells may be a factor contributing to nucleolin regulation, as cultured human leukemia-60 cells induced to differentiate into nonproliferating macrophages lose their ability to bind F3 (unpublished data). The restricted expression of cell surface nucleolin and the cell-type specificity of the expression may explain why some investigators have not been able to document the presence of nucleolin at the cell surface (Yu et al., 1998
). A similar explanation may apply to the heterogeneity of the cell surface nucleolin expression in the vasculature of tumors and matrigel plugs; local variation in endothelial cell proliferation is likely to occur in angiogenic lesions in vivo.
F3-displaying phage selectively homes to tumor vasculature in vivo, and fluorescein-tagged F3 also binds to and is taken up by endothelial cells in tumor vasculature. However, the peptide also spreads to tumor cells, and it appears in a few individual nonvascular cells in the skin and the gut (Porkka et al., 2002
). Intravenously injected antinucleolin antibody was only detected in angiogenic vessels of tumors as well as of matrigel plugs. The restricted distribution of the antibody resembles that of the phage, probably because the size of phage and antibody limit their access to tissues, whereas the relatively small molecular mass of the peptide conjugate (~5 kD) may permit wider distribution. Nonetheless, each of these reagents demonstrates the specificity of cell surface nucleolin for angiogenic vessels within the vasculature.
F3 is rich in basic amino acids and binds to cell surface heparan sulfate. However, our demonstration that CHO cells lacking heparan sulfate (and other glycosaminoglycans) internalize F3 excludes a direct role of heparan sulfate as the internalizing molecule. Indeed, binding and antibody inhibition studies show that F3 internalization is mediated by cell surface nucleolin. El-Sheikh et al. (2002)
have described a peptide from the heparin-binding domain of vascular endothelial growth factor that selectively homes to tumor vasculature. The authors attributed the tumor homing to affinity of the peptide for heparan sulfate. It will be interesting to see whether this peptide might also bind to nucleolin.
The internalization of the F3 peptide and NCL3 antibody may reflect a physiological function of cell surface nucleolin. Midkine is a 13-kD cytokine that, like F3, contains a high proportion of basic amino acids (Said et al., 2002
). It plays a role in neurite outgrowth and neuronal differentiation, and its mRNA is up-regulated in several human carcinomas (Tsutsui et al., 1993
). The internalization of midkine by cells has been reported to be nucleolin dependent (Said et al., 2002
), although lipoprotein receptor-related protein can also serve as the internalizing receptor for midkine (Shibata et al., 2002
). The binding site for midkine in nucleolin has been localized to the RGG domain of nucleolin (Said et al., 2002
), whereas our antibody inhibition results implicate the domain rich in acidic amino acids as the binding site for F3. Cell surface nucleolin may also be involved in the activities of basic FGF, which has been shown to bind to nucleolin in nuclear extracts (Bonnet et al., 1996
). Thus, F3, midkine, and possibly basic FGF, might be internalized by a nucleolin-dependent mechanism, but distinct binding sites on nucleolin may exist to mediate the uptake.
A highly basic peptide derived from the HIV Tat protein also binds to cells and is internalized by them. The Tat peptide allows internalization of conjugated proteins and is commonly used as a cell-penetrating agent (Fawell et al., 1994
; Langel, 2002
). It is unlikely that the Tat peptide would use nucleolin for its internalization and nuclear transport. First, the internalization of Tat is independent of the cell type, even in vivo, whereas our results show that cell surface nucleolin is limited, it is expressed in angiogenic endothelium but not in the blood vessels in normal tissues. Second, treatment of cells with heparinase to remove heparan sulfates inhibits internalization of the Tat peptide (Suzuki et al., 2002
), whereas we found that lack of heparan sulfates did not affect F3 uptake. Third, Tat internalization is independent of temperature and does not require energy, and several other cell-penetrating peptides are similar to Tat in this regard (Langel, 2002
). In contrast, F3 uptake is blocked at 4°
C (Porkka et al., 2002
). Finally, our antibody inhibition data also suggest that Tat peptide internalization is independent of nucleolin because an antinucleolin antibody inhibited the uptake of F3 but not of the Tat peptide.
Our laboratory has recently described yet another type of a cell-penetrating peptide, LyP-1, which is also rich in basic amino acids (Laakkonen et al., 2002
). This peptide specifically homes to the endothelium of tumor lymphatics and the tumor cells in certain, but not all, tumors. The internalization of this peptide is not affected by antinucleolin antibodies (unpublished data). Thus, several different internalization mechanisms for basic peptides appear to exist, both universal and cell-type specific.
Cell-penetrating peptides rich in basic amino acids are transported into the nucleus after internalization (Langel, 2002
). This is also the case with F3 and LyP-1 (Laakkonen et al., 2002
; Porkka et al., 2002
). Nucleolin is thought to be responsible for the nuclear transport of midkine (Shibata et al., 2002
), and the same may be the case with F3. It is also possible that the multiple basic amino acids in F3 form one or more independent nuclear localization signals.
The selective in vivo homing of the two nucleolin-binding reagents, the F3 peptide and the NCL3 antibody, to angiogenic blood vessels establishes cell surface nucleolin as a new angiogenesis marker. Tumor blood vessels undergo angiogenesis (Hanahan and Folkman, 1996
) and have specific markers in common with other angiogenic vessels (Ruoslahti, 2002
). Future studies will determine whether cell surface nucleolin might play a role in angiogenesis, possibly by binding and internalizing growth factors such as midkine and bFGF. The restricted expression of cell surface nucleolin in angiogenic vessels and in tumor cells in vivo, and its ability to internalize molecules bound to it, make nucleolin an attractive potential target for the development of agents for vascular therapy of tumors.