According to the current literature, it appears that nuclear secreted factors and RTKs generally regulate gene transcription. Though initial studies depicted binding of nuclear RTKs and GFs to DNA, no structural DBD has ever been characterized in these molecules. Therefore, it is likely that they do not directly bind DNA, but rather interact with DNA binding partners, and that their transcriptional activity depends on interactions with other transcription factors such as specific transcription factors and general co-regulators. Recent studies also revealed potential roles in other nuclear processes such as DNA repair and RNA processing.
Interaction with transcriptional regulators
EGFR and EGF were initially shown to bind chromatin, and SwGF, to AT-rich DNA sequences [44, 45]. We now know that some nuclear RTKs of the EGFR/ErbB family specifically bind target promoters. For instance, nuclear EGFR binds to AT-rich sequences in the promoter of the Cyclin D1 gene and activates its transcription . Likewise, ErbB2/HER2 binds to a specific sequence called HAS (HER2 associated sequence). The promoters of the Cyclooxygenase2 (COX2), PRPK, MMP16 and DDX10 genes were identified as direct target promoters of nuclear ErbB2 . It is worth noting that positive correlations have been established between nuclear localization of EGFR/ErbB receptors and expression levels of target genes in tumors (see below), underlining the pathophysiological relevance of these results. Nuclear EGFR complexes with STAT3 and co-regulates transcription of the iNOS gene . In a similar manner, its interaction with E2F1 activates the expression of the b-Myb gene, which encodes an essential transcription factor for cell cycle progression . EGFR-E2F1 association with the b-Myb promoter is only detected during the G1/S phase transition . Interestingly, inhibition of major EGFR downstream pathways such as PI-3K and ERK does not significantly suppress the EGFR-induced b-Myb expression . Similar to nuclear EGFR, ErbB4/HER4 co-activates the expression of the β-casein gene by binding STAT5a . ErbB4 may therefore potentiate the expression of milk genes by STAT5a during pregnancy and lactation. Similarly, nuclear PRL-CypB and GHBP potentiate STAT5 transcriptional activities [37, 50].
As for the EGF/ErbBs family, members of the FGFs/FGFRs family regulate the transcription of specific target gene by interacting with various nuclear partners. FGFR1 transcriptional activities were suggested when immunoelectron microscopic, and confocal analysis revealed that FGFR1 co-localized with transcriptionally active chromatin . FGFR1 binds to the general co-activator CREB-binding protein (CBP) and up-regulates the expression of specific target genes such as FGF2 and Tyrosine Hydroxylase by increasing the recruitment of RNA polymerase II and histone acetylation at active promoters [51, 52]. Nuclear FGFR1 also physically interacts with Ribosomal S6 Kinase isoform 1 (RSK1), a regulator of CBP and histone phosphorylation, and regulates its transcription activities toward transcriptional regulators complexes . The Neurofilament-L, neuron-specific enolase microtubule associated protein-2 (MAP2) and c-jun genes are now identified as target genes of nuclear FGFR1 [28, 51]). Nuclear FGFR1 also potentiates cyclin D1 expression . Nuclear FGFR1 thus regulates the expression of genes that are involved in cell growth and differentiation. Interestingly, as for EGFR, the transactivation of the target genes of nuclear FGFR1 is not induced by stimulation of cell-surface FGFR1, suggesting that the functions of a RTK at the plasma membrane may differ from the ones inside the nucleus.
Initially, externally added FGF2, which accumulated in the nucleus, was shown to correlate with stimulation of ribosomal gene transcription, and activate transcription in cell-free system [54–56]. More recently, in GST pull-down assays, nucleolin, histone H1, Upstream binding factor (UBF), an essential transcription factor for rRNA transcription, and ribosomal protein P0 were found as LMW FGF2 interacting partners . Furthermore, LMW FGF2 bound to UBF associates with rRNA genes and regulates rRNA transcription both in vitro and in vivo . Taken together, these results suggest a major role of nuclear LMW FGF2 in the regulation of rRNA genes. In addition, nuclear FGF2s regulate the transcription of genes transcribed in mRNAs. Indeed, the phosphoglycerate kinase 1 and 2 genes are regulated by nuclear FGF2 in a promoter-specific manner . Exogenously added LMW FGF2 enter the nucleus and directly interacts with RSK2 , another isoform of RSK, in a cell cycle-dependent manner. Nuclear LMW FGF2 may therefore potentiate the RSK2 activities during the cell-cycle progression (see below). Nuclear HMW FGF2s regulate the promoter activity of the IL-6 gene , and as FGFR1, they stimulate the transcription of the Tyrosine Hydroxylase gene via cAMP Response Element (CRE) sequences . HMW nuclear forms of FGF2 were also shown to physically interact with the anti-apoptotic putative transcription factor FIF2 . This interaction may be important for cell response to stress.
Preliminary data suggested that nuclear CCN proteins may regulate transcription. CCN2 activated transcription in a cell-free system , and the CCN3 CT module was found bound to a specific NFκB-like sequence in the promoter of the Plasminogen Activator Inhibitor-2 (PAI-2) gene . Intriguingly, no functional activity for this binding has been identified to date. We were unable to detect any variation of the transcription level of the luciferase gene cloned downstream human PAI-2 promoter sequences when co-transfected with various nuclear forms of CCN3 (data not shown and ). However, we found that CCN3 was able to modulate transcription in other systems. We used a single hybrid system in yeast, in which plasmids expressing CCN3 recombinant proteins fused to the DBD of the yeast Gal4 transcription factor. The recipient yeast used for transient transfections contained the lacZ reporter gene cloned downstream a promoter containing Gal4-DBD binding sites. The results reported in Figure 3B (Li et al. unpublished) indicated that under conditions in which the full-length CCN3 protein showed no activity, a CCN3 recombinant protein containing only IGFBP, VWC, and TSP1 modules (NH24) (nomenclature depicted in Figure 3A), induced a strong transcription transactivation. Similar results were obtained with pACT2-derived constructs, which are known to express higher levels of recombinant protein than pGADGH. The use of plasmids expressing either individual domains or combinations of domains, allowed us to establish that the VWC module of CCN3 was responsible for the transactivation activity of the recombinant proteins. Indeed, clones containing the VWC module (NH3, NH23, and NH24) were positive in the β-galactosidase assay, whereas NH2, NH4, NH5 and NH45 were negative. The negative results observed with NH35 (containing the VWC, TSP1 and CT modules) and with the full-length CCN3 (NH25) raised the possibility that the presence of the CT module was interfering with the transactivating activity of the VWC module. These results are in agreement with the transinhibitory effect of the CT module that we have described in a recent report .
In parallel, quantitative assays were performed in mammalian cells that confirmed the previous results obtained in yeast. We used a pFA-CMV plasmid that expressed the VWC module of CCN3 fused in frame with the DBD of Gal4 (see  for the construction strategy). A six histidine tag (6HIS) was added at the C-terminal part of the fusion protein to allow immunological detection of the recombinant proteins (Figure 3C, lower panel left). For the transactivation assays, cells were co-transfected with the pFA-NH3 expression plasmid, the pFR-Luc reporter plasmid, in which the luciferase gene transcription was under the control of 5 Gal4 binding sites, and a lacZ vector for β-galactosidase normalization. The results established that the VWC module could transactivate transcription in mammalian cells in a dose-response manner (Figure 3C, lower panel right). TADs are generally rich either in basic amino acids, or in proline. CCN3 VWC module encompasses both several basic and proline residues (Figure 3A). The IGFBP and TSP1 modules exert no significant effect on transcription (not shown), whereas the CT module contains an inhibitory domain of transcription that is stronger than the transactivation domain located in the VWC module . CCN3 nuclear forms may thus act as transcriptional repressors . Therefore, using a Gal4 reporter system, we showed that nuclear CCN3 forms were able to modulate transcription in various eukaryotic cells.
Interestingly, a similar situation has just been reported for IGFBP proteins. Indeed, a strong transactivation domain (TAD) was identified in the N-terminal part of IGFBP2, IGFBP3 and IGFBP5 using a similar method , but this TAD is masked by inhibitory domains located in the central and carboxy-terminal parts of the protein . IGFBP3 directly interacts with the Nuclear Retinoid X Receptor (RXR) and was shown to regulate gene transcription, using a RXR signaling reporter system [63, 64]. IGFBP3 was indeed able to activate transcription via the RXR-binding element (RXRE) and down-regulate transcription via RAR-binding element (RARE), suggesting a co-activator/repressor role for IGFBP3 in transcription . IGFBP3 interactions with RXR may lead to apoptosis in certain conditions (see below).
Regulation of transcription by nuclear RTKs was also initially investigated using such single hybrid systems . To date, functional TADs were identified for various RTKs such as EGFR, ErbB2/HER2 and ErbB4/HER4 [39, 46, 47], and it appears that nuclear GFs and RTKs regulate the transcription of specific target genes. These observations open interesting fields of investigation for the CCN proteins. At the plasma membrane, CCN proteins are described as docking proteins [7, 11, 12]. Similarly, in the nucleus, CCN proteins may act as co-regulators, forming bridges between transcription factors and the basal transcription machinery. Along this line, the rpb7 subunit of RNA polymerase II has been identified as a CCN3 partner in a two-hybrid assay in yeast .
In summary, the above examples depict the variety of mechanisms of action of nuclear GFs and RTKs on the regulation of transcription. Some of them (such as the EGF/ErbB family, PRL, nucleolar FGF2) activate the transcription of specific target genes by interacting with specific transcription factors. Molecules like FGF1 and FGF2 can also interact with general co-regulators such as CBP and RSK proteins. Others (such as IGFBPs) seem to be able to either activate or inhibit gene transcription depending on their binding partners. They contain both TADs and TIDs (transinhibitory domains).
Other actions in the nucleus
In addition to transcription, secreted proteins and RTKs that are transported to the nucleus seem also to directly regulate other important nuclear processes such as DNA repair and RNAs processing. Promoting DNA repair by ionization induces nuclear translocation of EGFR. This translocation results in activation of DNA-dependent Protein Kinase (DNA-PK), an important effector for the repair of DNA double-strand breaks . These results suggest a role for nuclear EGFR in DNA repair and cell survival after irradiation.
Several studies suggest functions in the metabolism of the different RNAs. For example, PTHrP has been detected in the nucleus/nucleolus of a large variety of cells. PTHrP contains in its N-terminal part a consensus sequence found in numerous RNA binding proteins. PTHrP was found to directly bind several types of RNAs, suggesting a role in RNA metabolism .
Along the same line, nuclear FGF3 physically interacts with the Nucleolar FGF3-Binding Protein (NoBP; a.k.a. Ebp2p) and ribosomal protein S2 (rpS2) [68, 69]. NoBP/Ebp2p is required for pre-rRNA processing and is essential for the synthesis of the 60S ribosomal subunit. RpS2 is a component of the small sub-unit of the ribosome. Nuclear FGF3 may therefore regulate ribosomal biogenesis.
HMW FGF2 (23 kDa) is associated with small nuclear Ribonucleoproteins particles (snRNPs) by physical interaction with the Survival of Motoneuron (SMN) protein, that functions as an assembly and recycling factor for the splicing machinery . SMN can be located in the cytoplasm and in the nucleus. HMW FGF2–23 co-localises with SMN in the nucleus . One of the subunits of the splicing factor 3a was identified as a binding partner for HMW FGF2–23 . Electron microscopy studies revealed co-localization of FGFR1 with snRNPs in the nucleus of differentiating neurons but not proliferating neurons in cultures . Therefore, nuclear FGF2/FGFR1 may be involved in mRNA processing during neuronal differentiation. Moreover, in as much as FGF2 is a neurotrophic factor for motoneurons (see below) and as SMN is deficient in patients with spinal muscular atrophy, a neurodegenerative disease, the FGF2-SMN complexes may be important for survival or degeneration of motoneurons.
The nuclear activities of secreted factors and RTKs cited above are summarized in Figure 2.