Published online before print March 4, 2004, doi: 10.1161/01.RES.0000124761.62846.DF
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© 2004 American Heart Association, Inc.
UltraRapid Communication |
Evidence That IGF Binding Protein-5 Functions as a Ligand-Independent Transcriptional Regulator in Vascular Smooth Muscle Cells
From the Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Mich.
Correspondence to Cunming Duan, Department of MCDB, University of Michigan, Natural Science Building, Room 3065B, Ann Arbor, MI 48109-1048. E-mail cduan{at}umich.edu
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Abstract |
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Insulin-like growth factor binding protein (IGFBP)-5 is a conserved protein synthesized and secreted by vascular smooth muscle cells (VSMCs). IGFBP-5 binds to extracellular IGFs and modulates IGF actions in regulating VSMC proliferation, migration, and survival. IGFBP-5 also stimulates VSMC migration through an IGF-independent mechanism, but the molecular basis underlying this ligand-independent action is unknown. In this study, we show that endogenous IGFBP-5 or transiently expressed IGFBP-5-EGFP, but not IGFBP-4-EGFP, is localized in the nuclei of VSMCs. Using a series of IGFBP-4/5 chimeras and IGFBP-5 points mutants, we demonstrated that the IGFBP-5 C-domain is necessary and sufficient for its nuclear localization, and residues K206, K208, K217, and K218 are particularly critical. Intriguingly, inhibition of protein secretion abolishes IGFBP-5 nuclear localization, suggesting the nuclear IGFBP-5 is derived from the secreted protein. When added exogenously, 125I- or Cy3-labeled IGFBP-5 is capable of cellular entry and nuclear translocation. To identify potential transcriptional factor(s) that interact with IGFBP-5, a human aorta cDNA library was screened by a yeast two-hybrid screening strategy. Although this screen identified many extracellular and cytosolic proteins that are known to interact with IGFBP-5, no known transcription factors were found. Further motif analysis revealed that the IGFBP-5 N-domain contains a putative transactivation domain. When fused to GAL-4 DNA dinging domain and tested, the IGFBP-5 N-domain has strong transactivation activity. Mutation of the IGF binding domain or treatment of cells with IGF-I has little effect on transactivation activity. These results suggest that IGFBP-5 is localized in VSMC nucleus and possesses transcription-regulatory activity that is IGF independent. The full text of this article is available online at http://circres.ahajournals.org.
Key Words: insulin-like growth factor-1 • nuclear localization signal • vascular smooth muscle cells • transcription factors
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Introduction |
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There is an accumulating body of evidence that locally produced insulin-like growth factors (IGFs) may play a critical role in the development and progression of atherosclerotic plaques.1–5 Most, if not all, IGFs in the extracellular environments are bound to specific, high-affinity IGF binding proteins (IGFBPs). IGFBPs are a family of six secreted proteins that bind IGFs and modulate IGF distribution, stability, and biological activities.6,7 Previous studies have shown that mammalian VSMCs synthesize and secrete IGFBP-2, -3, -4, and -5.1,2,8 These IGFBPs can inhibit or potentiate IGF-I–induced VSMC proliferation and migration.9–14 Recently, we have shown that these locally produced IGFBPs also play an important role in determining whether VSMCs proliferate or migrate in responses to IGF-I stimulation and a key player in this paradigm is IGFBP-5.8,14 IGFBP-5 not only modulates IGF-I actions, but it also directly stimulates VSMC migration through an IGF-independent mechanism(s).14 The molecular basis underlying the ligand-independent action of IGFBP-5 is unknown.
Ligand-independent actions of IGFBP-5 on cell growth and differentiation have been documented in human osteoblast cells and breast cancer cells,15–19 although the underlying molecular mechanism(s) remains poorly understood. In human osteoblast cells, IGFBP-5 has been reported to bind to a putative cell surface receptor,20 but the molecular identity of this IGFBP-5 "receptor" is still unknown. Recognizing that multiple basic residues in the 201 to 218 region of IGFBP-5 are similar to the bipartite nuclear localization signal (NLS) found in viral and mammalian transcription factors, it was speculated that IGFBP-5 might also act in an "intracrine" fashion, perhaps serving as a cytosol-to-nuclear shuttle for its ligand.21 Indeed, when added to cultured human bone tumor and breast cancer cells, exogenous IGFBP-5 was shown to be capable of cellular and nuclear entry.22,23 Likewise, when fused to EGFP and transfected into CHO cells, a peptide corresponding to the NLS of IGFBP-5 (residues 201 to 218) targeted EGFP to the nucleus.24 However, it is not clear whether endogenous IGFBP-5 is present in the nucleus in VSMCs or any other cell types, nor is understood where is the nuclear IGFBP-5 is derived from. Moreover, the functional significance of IGFBP-5 in the nucleus and its role, if any, in mediating the IGF-independent actions of IGFBP-5 are unknown in VSMCs or any other cell types.
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Materials and Methods |
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Materials
All chemicals and reagents were purchased from Fisher Scientific unless noted otherwise. The IGFBP-5 antibodies were purchased from Upstate Biotechnology Inc and Diagnostic Systems Laboratories, Inc. The C-Fos and IGF-IR antibodies were purchased from Santa Cruz Biotech, and Akt antibody was obtained from Cell Signaling Inc. Fetal bovine serum (FBS), cell culture media, antibiotics, and trypsin were purchased from GIBCO/BRL. Peptides were synthesized at the Protein Structure Facility, University of Michigan. Peptide BP5(201-218) corresponds to IGFBP-5 amino acid residues Arg201-Arg218 and peptide BP5(131-141) peptide includes IGFBP-5 amino acid residues Ala131-Thr141. They were purified by HPLC to >90% purity and analyzed by mass spectroscopy before use.
Cell Culture, Subcellular Fractionation, Immunological Assays, and Ligand Blot
Porcine VSMCs were isolated and cultured as reported previously.25 Human embryonic kidney (HEK) 293, A7R5, and CHO-K1 cells were obtained from ATCC and were cultured as recommended. For immunostaining, cells were grown on 8-chamber slides (Falcon). After permeabilization and blocking in 3% BSA/1% Triton X-100/PBS, they were incubated with the primary antibody at 4°C overnight. After washing, the cells were incubated with appropriate secondary antibodies and microphotographs were taken with a Nikon EC600 Fluorescence Microscope. Cells were fractionated following a previously published method.26 These fractions were processed to immunoprecipitation, immunoblot, and ligand blot analysis as reported previously.14
Plasmid Construction
For the construction of IGFBP-EGFP expression plasmids, DNA fragments corresponding to the signal peptide plus N-domains (containing residues -21 to 79 of IGFBP-4 and residues -20 to 80 of IGFBP-5), L-domains (containing residues 80 to 150 and 81 to 169 of IGFBP-4 and -5, respectively), C-domains (containing residues 151 to 237 and 170 to 252 of IGFBP-4 and -5), NL-domains, and LC-domains of human IGFBP-4 and -5 were generated by PCR amplification as described previously.27 DNA fragments encoding various IGFBP-5 point mutants were generated by PCR using mutant DNA provided by Dr D.R. Clemmons, University of North Carolina at Chapel Hill. The fragments were subcloned into the HindIII/KpnI or XhoI/KpnI sites of pEGFP-N1 vector (Clontech). The DNAs were transfected into cells using LipofectAMINE method.14 The transfected cells were washed and photographed. To produce Gla4-DBD and IGFBP fusion proteins, DNA fragments corresponding to the N-domain of human and zebrafish IGFBP-5 and -1 were generated by PCR and subcloned into the EcoRV/XbaI sites of pBIND vector (Promega) in frame. All plasmids were confirmed by DNA sequencing.
Yeast Two-Hybrid Screen
The screen was performed as reported recently.27 Briefly, the Matchmaker two-hybrid system 3 (CLONTECH) was used to identify candidate proteins that interact with IGFBP-5. The bait, pGBKT7-IGFBP-5, generated by inserting full-length human IGFBP-5 cDNA into the NcoI and BamH1 sites of the pGBKT7, was used to screen a human aorta cDNA library constructed in the pACT2 vector (CLONTECH). Positive clones were identified, and they were retested twice under high stringency.
Internalization Assays
The internalization of 125I-labeled IGFBP-5 was performed following previously published method.15,26 Peptide BP5(201-218) was labeled with activated Cy3 dye (Amersham Pharmacia Biotech) following the manufacturer’s protocol. The labeled peptide was purified by a Sephadex G-25 column and was eluted with 1x PBS (pH7.4). The collected fractions were analyzed by spectrophotometer at wavelengths of 280 and 552 nm and the labeled peptide was quantified using a standard curve of the pure peptide. The calculated dye/peptide ratio was about 0.4. For the internalization assay, cells were grown in the 4-well or 8-well chamber slides (Nalge Nubc Intl). After washing with serum-free medium (SFM) containing 0.1% BSA three times, cells were incubated with Cy3-labeled BP5(201-218) (2.5 µg/mL) or Cy3-IgG ( 8.0 µg/mL) in SFM containing 0.1% BSA for 4 hours at 37°C. After incubation, the cells were washed twice with 2 mol/L NaCl in 1x PBS followed by further washings with 1x PBS. The cells were then fixed with 4% paraformaldehyde, stained with DAPI, and examined under a fluorescence microscope. The internalization of IGFBP-5-EGFP was performed under similar conditions.
One-Hybrid Transcription Assay
The transcription activation activity of IGFBP-5 N domain was determined following Kabuta et al.28 Briefly, cells were cotransfected with pG5-luc and each pBIND-IGFBP fusion construct. Twenty-four hours after transfection, cells were washed and lysed. The lysates were measured for firefly and Renilla luciferase activities using the Dual-Luciferase Reporter assay system (Promega). The result was expressed as fold change over the pBIND empty vector group. Transfection efficiency was normalized by Renilla luciferase activity.
Statistical Analysis
Values are mean±SE. Differences among groups were analyzed by one-way analysis of variance followed by Fisher’s protected least significance difference test using Stat-View (Abacus Concept, Inc).
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Results |
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IGFBP-5 Is a Nuclear Protein in VSMCs
To determine whether endogenous IGFBP-5 is present in the nuclei of VSMCs, primarily cultured porcine VSMCs were subjected to immunocytochemical staining using a polyclonal and a monoclonal IGFBP-5 antibody. These antibodies do not cross-react with other IGFBPs.10,14 As shown in Figure 1A, IGFBP-5 was detected in the nuclei by both antibodies (Figures 1Aa and 1Ac). Preabsorption of the IGFBP-5 antibodies by excess IGFBP-5 abolished the nuclear IGFBP-5 signal (Figure 1Ad), confirming the authenticity of the antibody staining. Figure 1Ad shows an overexposed view in order to appreciate the complete lack of nuclear signal. As a control, VSMCs were stained with a c-Fos (a nuclear protein) antibody and an IGF-IR (a transmembrane receptor kinase) antibody (Figures 1Ae and 1Af). Next, cells were fractionated into cytosolic, membrane, and nuclear fractions. These fractions were precipitated by the IGFBP-5 antibody and analyzed by ligand blot using 125I-IGF-I. A 30-kDa protein in the nuclear and membrane fractions was precipitated by the IGFBP-5 antibody, and this protein was capable of 125I-IGF-I binding (Figure 1B). These fractions were also subjected to immunoblot using antibodies against IGF-IR, Akt, and c-Fos. IGF-IR was detected in the membrane fraction, whereas Akt was seen predominantly in the cytoplasmic faction (Figure 1B). c-Fos, on the other hand, was exclusively detected in the nuclear fraction, suggesting that these fractions were relatively clean. We further investigated the nuclear localization of IGFBP-5 by transiently expressing an IGFBP-5-EGFP fusion protein and observing the EGFP signal in living cells. EGFP and IGFBP-4-EGFP were used as controls. EGFP is a small protein that can move between the nuclear and cytoplasmic compartments. When transfected to VSMCs, EGFP was detected in both the cytoplasm and nucleus (Figures 1Ca and 1Cb). The IGFBP-5-EGFP signal, on the other hand, was seen predominantly in the nucleus (Figures 1Cc and 1Cd). In comparison, the IGFBP-4-EGFP was not seen in the nucleus (Figures 1Ce and 1Cf). Similar results were obtained in A7R5 cells, a transformed cell line derived from rabbit aorta (Figure 1Cg and 1Ch). The easily transfectable A7R5 cells were therefore used in subsequent experiments. Data from five independent experiments indicated 83±2% of the IGFBP-5-EGFP–transfected cells showed exclusive nuclear presence, whereas none of the IGFBP-4-EGFP–transfected cells showed nuclear signal.
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Several Basic Residues in IGFBP-5 C-Domain Are Critical for IGFBP-5 Nuclear Localization
IGFBP-5 and IGFBP-4 share similar domain structure, substantial sequence identity, and the ability to bind IGFs with high affinities, but only IGFBP-5 is localized in the nucleus. We therefore used a strategy based on IGFBP-4/5 chimeras to map the unique region in IGFBP-5 responsible for its nuclear localization. Six IGFBP chimeras, namely, IGFBP-545, -454, -554, -445, -544, and -455, were generated by exchanging the N-, L-, and C-domain of the two IGFBPs (Figure 2A). Ligand blot analysis revealed that all six chimera proteins retained IGF binding ability (data not shown). The six IGFBP chimeras were fused to EGFP and introduced into A7R5 cells by transient transfection. As shown in Figure 2B, the three chimeras lacking the IGFBP-5 C-domain, namely IGFBP-544, -454, and -554, exhibited little or no nuclear presence (0%, 3.6±1.2%, and 4.8±1.2%, respectively). In contrast, IGFBP-455, -445, and -545, all containing the IGFBP-5 C-domain, were predominantly nuclear (88±8.4%, 76±3.6%, and 65±1.2%, respectively), indicating that the IGFBP-5 C-domain is necessary and sufficient for its nuclear localization.
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The human IGFBP-5 C-domain contains a putative NLS motif between residues 201 to 218. This sequence is perfectly conserved in all known vertebrate IGFBP-5 homologues ranging from human to zebrafish, but is absent in IGFBP-4. Mutation of K217/R218 into 217A/218A reduced the nuclear presence by 72% (Figure 2C). Mutation of two additional basic residues in this half, ie, changing K211/R214/K217/R218 into 211N/214A/217A/218A, resulted in an 82% reduction. Changing R201/K202 into A201/N202 caused a modest 17% decrease. Further mutation of K206/K208 into N206/N208 on the A201/N202 background resulted in a 66% reduction (Figure 2C). These data suggest that K206, K208, K217, and K218 are critical. To prove that the particular sequence in the NLS motif of IGFBP-5, but not the charge changes introduced by these mutations, is critical, four basic residues in the L-domain, K133/R136/K138/K139, were changed into N133/A136/A138/A139. This mutant acted as the wild-type protein (Figure 2C).
Nuclear IGFBP-5 Is Derived From Secreted Protein
To show that the IGFBP-5-EGFP fusion protein is secreted and retains IGF binding property and to determine whether the nuclear IGFBP-5 is derived from the secreted protein, stable CHO-K1 cell lines expressing IGFBP-5-EGFP were obtained. Analysis of the media conditioned by the transfected cells indicated the presence of a secreted protein at the predicted size (55 kDa). We conclude this to be IGFBP-5-EGFP because it bound 125I-IGF-I and was immunoreactive to a GFP antibody (Figure 3A). When brefeldin A was added, IGFBP-5-EGFP disappeared from the conditioned media (Figure 3B), confirming that the IGFBP-5-EGFP detected was originated from secretion. Intriguingly, the inhibition of IGFBP-5-EGFP secretion abolished the nuclear localization of IGFBP-5-EGFP, whereas it increased its cytoplasmic presence (Figures 3Ca and 3Cc). This suggests that the nuclear IGFBP-5 is not simply imported from cytoplasm but derived from the secreted protein.
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To test whether the secreted IGFBP-5 could enter into the cells and subsequently into the nucleus, 125I-iodine labeled human IGFBP-5 was added to cultured VSMCs. 125I-IGFBP-5 was rapidly internalized by VSMCs and reached maximal levels 4 hours after the addition (Figure 4A). In the presence of excess amounts of unlabeled IGFBP-5, but not IGFBP-4, the internalization was significantly reduced (Figure 4B), suggesting that this is a specific event. Likewise, a peptide corresponding to the NLS region of IGFBP-5, peptide BP5(201-218), was translocated into the nucleus with a similar time course (Figures 4Ca and 4Ce). The unclear uptake was abolished when excess amount of unlabeled BP5(201-218) was added (Figures 4Cb and 4Cf). Addition of BP5(131-141), another basic peptide corresponding to residues 131 to 141 of human IGFBP-5, had no such effect (Figures 4Cc and 4Cg). To show that full-length IGFBP-5 is capable of nuclear entry, recombinant IGFBP-5-EGFP was generated. When added to cultured cells, the GFP signal was detected in the nucleus after 6 hours (Figures 4Da and 4Db).
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IGFBP-5 N-Domain Possesses Transactivation Activity That Is Evolutionarily Conserved
The nuclear localization of IGFBP-5 raised the possibility that it may regulate gene expression directly or indirectly. To identify potential IGFBP-5 interacting proteins, we recently screened a human aorta cDNA library using a yeast two-hybrid screening strategy. Over 60 positive clones were obtained after screening 4.3 million colonies using human IGFBP-5 as bait. As reported in a recent article, 28 of these encode polypeptides corresponding to fibronectin.27 Others encoded extracellular and cytosolic proteins such as fibrin and importin, which are known to interact with IGFBP-5.6,7 No known transcription factors, however, were found by this approach.
By motif analysis, we found that the IGFBP-5 N-domain contains a conserved proline-rich sequence, which is a typical feature of transactivation domains of transcription factors. To determine whether the IGFBP-5 N-domain has any transactivation activity, human IGFBP-5 N-domain was fused to the Gal4-DNA binding domain (DBD) and introduced to VSMCs together with a Gal4 reporter plasmid. As shown in Figure 5A, IGFBP-5 N-domain caused a highly significant increase in Gal-4 reporter gene transcription over the Gal4-DBD control (Figure 5A). An even greater activity (up to 33-fold increase) was observed when tested in human HEK293 cells (Figure 5B). This was probably due to the higher transfection efficiency in HEK293 cells. The transactivation activity is unique to IGFBP-5 N-domain and is evolutionarily conserved. Human IGFBP-1 N-domain had no such activity. Likewise, the N-domain of zebrafish IGFBP-5, but not zebrafish IGFBP-1, caused a significant increase when tested in HEK293 cells although the activity of the fish protein was considerably weaker in these human cells (Figure 5C).
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Nuclear Activity of IGFBP-5 Is Ligand Independent
Because the IGFBP-5 N-domain also contains the high-affinity IGF binding site,29,30 we wondered whether the transactivation activity of IGFBP-5 is ligand dependent. Nuclear uptake of IGF-I has been reported in other cell types.31 We therefore examined the effect of IGF-I treatment. The addition of IGF-I (200 ng/mL) did not alter the transactivation activity (Figure 6A). Next, the activity of mini-IGFBP-5, which comprises residues 40 to 92 of human IGFBP-5, was determined. Mini-IGFBP-5 contains the high-affinity IGF binding site and can bind to IGFs at nanomolar concentrations.29 As shown in Figure 6B, mini-IGFBP-5 had no transactivation activity. In contrast, a construct covering residues 1 to 39 of human IGFBP-5 N-domain (BP5N1-39) had significant transactivation activity (Figure 6B). We further compared the activity of the N-domain of a non-IGF binding IGFBP-5 mutant and wild-type IGFBP-5. This mutant (K68N/P69Q/L70Q/L73Q/L74Q) has a 1000-fold reduced affinity for IGF-I.30 As shown in Figure 6C, it had similar transactivation activity as the wild-type IGFBP-5 (Figure 6C). Taken together, these data indicate that the transactivation activity of IGFBP-5 is ligand independent and the transactivation domain is distinct from the IGF binding domain.
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Discussion |
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In this study, we have demonstrated that IGFBP-5 is not only secreted but also localized in the nucleus in VSMCs. The nuclear IGFBP-5 has a molecular mass similar to that of the secreted protein and is capable of IGF binding. We have provided several lines of evidence indicating that the nuclear IGFBP-5 is derived from the secreted protein. Our study further reveals the presence of an evolutionarily conserved, functional transactivation domain in IGFBP-5 N-domain. This novel activity of IGFBP-5 is IGF independent and can be physically separated from the IGF binding site. The nuclear localization and nuclear action of IGFBP-5 provides a molecular basis for several prominent IGFBP-5 activities that are IGF independent.
Although the presence of a putative NLS in IGFBP-5 C-domain was previously suggested,21 and exogenous IGFBP-5 was found to be capable of nuclear uptake by cultured human breast and bone tumor cells22,23; to our knowledge, the present study is the first report demonstrating that endogenous IGFBP-5 is localized in VSMC nucleus. This conclusion was supported by several independent experimental approaches, including (1) colocalizing IGFBP-5 with DNA by immunocytochemistry using two independent antibodies, (2) cell fractionation followed with immunoprecipitation and ligand blot analysis, and (3) transfecting VSMCs with IGFBP-5-EGFP fusion constructs. Using a series of IGFBP-4/5 chimeras and IGFBP-5 points mutants, we show that the IGFBP-5 C-domain is both necessary and sufficient for its nuclear localization, and residues K206, K208, K217, and K218 are particularly critical. This is in overall agreement with a previous study in which K206/R207/K208 and R214/G215/R216/K217/R218 were found to be important.22 However, there are key differences between the two studies. In the study by Schedlich et al,22 a peptide corresponding to 201 to 218 of IGFBP-5 was fused to EGFP. The resulted fusion protein is 27 kDa and as such is capable of passive diffusions into the nucleus.32 More importantly, this 27-kDa fusion protein is not a functional IGFBP. The EGFP fusion protein used in our study was generated by fusing EGFP to the C-terminus of the entire coding region of human IGFBP-5. The resulted fusion protein is 
55 kDa and therefore cannot enter the nucleus by diffusion. Furthermore, our IGFBP-5-EGFP is secreted and capable of IGF binding.
There are several plausible sources/routes for the nuclear IGFBP-5. First, an intracellular isoform(s) of IGFBP-5 may exist, and it is translocated into the nucleus under the guidance of the NLS. Such a mechanism has been documented for fibroblast growth factor (FGF)-2 and parathyroid hormone related peptide (PTHrP).33,34 In the case of FGF-2, alternative initiation of translation of FGF2 mRNA occurs at three CUG codons downstream of the conventional AUG start codon.33 These CUG-initiated variants of FGF-2s were shown to remain in the cytoplasm and translocate to the nucleus. Retrograde trafficking from ER to the nucleus is another plausible mechanism of translocating the intracellular IGFBP-5 from cytoplasm to the nuclear compartment. It has been suggested that misfolded proteins that would classically undergo degradation in the ER can be back-translocated from the ER to the cytoplasm.35 Finally, the secreted IGFBP-5 may reenter the cell by an endocytosis-dependent pathway. We favor the last model for following reasons: (1) the endogenous IGFBP-5 in the nucleus is the mature protein capable of IGF binding; (2) IGFBP-5-EGFP is secreted as well as localized in the nucleus; (3) exogenously added, functional IGFBP-5 is capable of cell entry and nuclear translocation with relatively rapid kinetics; and (4) inhibition of protein secretion by BFA abolishes the nuclear presence of IGFBP-5-EGFP, whereas it increases its cytoplasmic content. This conclusion is in good agreement with a recent report that IGFBP-3, a closely related member of the IGFBP family, can be internalized through transferrin receptor–mediated and caveolin-mediated endocytic pathways in 22RV1 and PC3 prostate cancer cells.36 The cellular receptor(s) mediated IGFBP-5 internalization is unknown at present. IGFBP-5 has been shown to interact with heparan sulfate (HS) proteoglycans located on VSMC surface, and this interaction is important for the ligand-independent action of IGFBP-5 on VSMC migration.14 Cell surface HS proteoglycans are known to act as cellular receptors for several viruses and viral protein, including HIV tat protein, adeno-associated virus type 2 virions, foot-and-mouth disease type O virus, HSV types 1 and 2, and dengue virus.37 They are also involved in the internalization and nuclear localization of a number of growth factors and cytokines such as fibroblast growth factor-2.38 A recent study indicated that HS proteoglycans themselves can be targeted into the nucleus of cultured primary corneal fibroblasts and this process is regulated by fibronectin.39 Toward this end, it is worthy noting that IGFBP-5 also interacts fibronectin and this binding is independent from IGF binding.27 This binding is mediated by IGFBP-5 C domain. This is consistent with the observation made in this study that IGFBP-5 C domain is critical for its internalization and nuclear localization. It will be of interest to determine whether the binding of IGFBP-5 with HS proteoglycans and/or fibronectin plays any role in IGFBP-5 internalization.
Another novel finding made in this study is that IGFBP-5 N-domain contains a functional transactivation domain. When fused to a heterogeneous DNA binding domain, the IGFBP-5 N-domain has strong transactivation activity. This activity is evolutionarily conserved and is not found in IGFBP-1 N-domain. Furthermore, this activity of IGFBP-5 is not affected by IGF-I binding nor by mutations in the ligand binding site. In addition, we were able to partially separate the transactivation domain from the IGF binding domain. These findings suggest that IGFBP-5 possesses transcription-regulating activity that is ligand-independent and may function as a transcriptional regulator or coregulator in VSMC nucleus in an IGF-independent fashion. It should be pointed out, however, that our results only prove that IGFBP-5 N-domain has the ability to activate the GAL-4 reporter gene when fused to GAL-4-DBD. More studies are needed to determine whether endogenous IGFBP-5 has similar activity and to identify its target genes.
Our findings suggest a novel pathway by which the secreted IGFBP-5 reenters cells and targets to the nuclear compartment where it may exert ligand-independent actions on gene expression. The concept that a secreted protein may enter the nucleus and exert nuclear actions is highly unusual but not without precedents. An increasing number of growth factors/cytokines with classical roles as extracellular signaling molecules and their cognate receptors are now found in the nucleus, where some of them are reported to act in an "intracrine" fashion.40,41 Recently, it was reported that secreted IGFBP-3, a closely related member of the IGFBP family, is internalized by prostate cancer cells through both transferrin receptor and caveolin-mediated endocytic pathways.36,42 It was reported that IGFBP-3 can interact with retinoid X receptor-
, and this interaction resulted in the modulation of the transcriptional activity of retinoid X receptor-.43 Despite previous reports on the nuclear presence of IGFBPs, their nuclear functions and the underlying mode of actions have not been vigorously explored. Our findings that IGFBP-5 is present in the nucleus of VSMCs and possesses ligand-independent transactivation activity have raised the possibility that IGFBP-5 may function as a hormone binding protein as well as a transcriptional regulator depending on its location. More studies are needed to identify the target genes of IGFBP-5 and to elucidate the molecular events that trigger its cell entry and nuclear translocation in VSMCs.
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Acknowledgments |
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This study was supported in part by NIH Grant RO1HL60679 and National Science Foundation Grant IBN 0110864 to C.D. We thank Dr David R. Clemmons, University of North Carolina at Chapel Hill, for providing the IGFBP-5 antibody and mutant IGFBP-5 cDNAs as PCR templates, Dr Shin-Ichiro Takahashi, University of Tokyo, for his help with the one-hybrid transcriptional assay, and Dr Antony Wood, University of Michigan, for critical reading of this manuscript.
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Footnotes |
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Original received January 22, 2004; revision received February 18, 2004; accepted February 20, 2004.
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References |
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