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Decreased caveolin-1 levels contribute to fibrosis and deposition of extracellular IGFBP-5

Abstract Our previous studies have demonstrated increased expression of insulin-like growth factor binding protein-5 (IGFBP-5) in fibrotic tissues and IGFBP-5 induction of extracellular matrix (ECM) components. The mechanism resulting in increased IGFBP-5 in the extracellular milieu of fibrotic fibroblasts is unknown. Since Caveolin-1 (Cav-1) has been implicated to play a role in membrane trafficking and signal transduction in tissue fibrosis, we examined the effect of Cav-1 on IGFBP-5 internalization, trafficking and secretion. We demonstrated that IGFBP-5 localized to lipid rafts in human lung fibroblasts and bound Cav-1. Cav-1 was detected in the nucleus in IGFBP-5-expressing fibroblasts, within aggregates enriched with IGFBP-5, suggesting a coordinate trafficking of IGFBP-5 and Cav-1 from the plasma membrane to the nucleus. This trafficking was dependent on Cav-1 as fibroblasts from Cav-1 null mice had increased extracellular IGFBP-5, and as fibroblasts in which Cav-1 was silenced or lipid raft structure was disrupted through cholesterol depletion also had defective IGFBP-5 internalization. Restoration of Cav-1 function through administration of Cav-1 scaffolding peptide dramatically increased IGFBP-5 uptake. Finally, we demonstrated that IGFBP-5 in the ECM protects fibronectin from proteolytic degradation. Taken together, our findings identify a novel role for Cav-1 in the internalization and nuclear trafficking of IGFBP-5. Decreased Cav-1 expression in fibrotic diseases likely leads to increased deposition of IGFBP-5 in the ECM with subsequent reduction in ECM degradation, thus identifying a mechanism by which reduced Cav-1 and increased IGFBP-5 concomitantly contribute to the perpetuation of fibrosis.


Introduction
Fibrotic disorders, including systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF), are characterized by excessive deposition of extracellular matrix (ECM) components such as collagen and fibronectin (FN) [1,2]. Fibrosis is the result of abnormal ECM remodelling which occurs due to an imbalance of ECM deposition and degradation [3,4]. Activated fibroblasts are a primary source of ECM in fibrotic tissues [5]. Although numerous studies have explored the fibrotic process in different tissues, the exact pathogenic mechanism responsible for fibrosis in SSc and IPF has not been elucidated.
Caveolin-1 (Cav-1) is a 22-kD membrane protein essential for the formation of flask-shaped 50-100 nm membrane invaginations termed caveolae [6]. Since caveolae have a distinctive composition of lipids, including cholesterol and sphingolipids, they serve as a platform for processes such as trafficking, endocytosis and signalling [7][8][9]. Cav-1 was recently implicated in fibrosis. Kasper et al. first reported abnormal Cav-1 expression in type I pneumocytes during lung fibrogenesis [10]. Tourkina et al. subsequently identified a role for Cav-1 in regulating collagen expression in lung fibroblasts [11]. In addition, markedly decreased expression of Cav-1 in primary fibroblasts, lung and skin tissues of patients with SSc and IPF was reported [12,13]. Several studies have shown that Cav-1 regulates a variety of signalling molecules and receptors, including Smad/transforming growth factor (TGF)-␤ receptor, Akt, extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated protein kinase (MAPK)/ERK (MEK), which interact with Cav-1 scaffolding domain (CSD) corresponding to amino acids 82-101 of Cav-1 [14][15][16]. Further confirmation for the role of Cav-1 in fibrosis was demonstrated in Cav-1 knockout mice, which exhibit a wide range of fibrosis-like lung abnormalities including thickening of lung alveolar septa and presence of hypertrophic type II pneumocytes [17,18].
Insulin-like growth factor binding proteins (IGFBPs) were originally reported as regulators of insulin-like growth factor (IGF)-I function [19]. Secreted IGFBPs serve as carriers of IGF-I and modulate IGF-I actions, either potentiating or inhibiting them. Several studies have reported that IGFBPs also exert IGF-I-independent effects [20][21][22][23]. IGFBP-3 and -5 are the most abundant and conserved IGFBPs, respectively. They are secreted proteins which can also translocate to the nucleus via a nuclear localization sequence [24,25]. Nuclear IGFBP-3 and -5 are believed to be derived from the corresponding secreted proteins, and their uptake by cells likely occurs through putative receptors which have not yet been identified [26].
We have previously reported that IGFBP-5 is significantly increased in dermal and pulmonary fibroblasts and tissues of patients with SSc and IPF [27,28]. IGFBP-5 binds ECM components and its deposition in the extracellular milieu is increased in fibrotic cells and tissues [28]. Notably, IGFBP-5 induces the production of ECM components in vitro by IGF-I independent mechanisms [29,30] and triggers a fibrotic phenotype in vivo [31] that includes induction of ECM production, myofibroblastic transformation and infiltration of mononuclear cells [29,32]. Furthermore, IGFBP-5 triggers significant fibrosis in human skin using an ex vivo organ culture model [33].
In this study, we investigated the internalization and nuclear translocation of IGFBP-5 in association with Cav-1 in primary lung fibroblasts. Our findings demonstrate that IGFBP-5 binds Cav-1, is internalized via Cav-1-mediated pathways, and subsequently translocates to the nucleus as vesicular-like aggregates that lack a membrane and contain Cav-1. Further, IGFBP-5 is increased in the ECM of fibroblasts from Cav-1 null mice and likely protects ECM components from degradation. Thus, in fibrotic disorders such as SSc and IPF, reduced Cav-1 levels result in increased extracellular levels of IGFBP-5, which then contributes to fibrosis both by inducing ECM production and protecting ECM components from degradation.

Primary fibroblast culture
Human primary lung fibroblasts were cultured under a protocol approved by the University of Pittsburgh Institutional Review Board from the explanted lungs of normal organ donors. Mouse primary lung fibroblasts were cultured from lung tissues of C57BL/6J wild-type (WT) mice and Cav-1 null mice (Cav-1 -/-) (The Jackson Laboratory, Bar Harbor, ME, USA). Approximately 2 cm pieces of peripheral lung were minced and fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; Mediatech, Herndon, VA, USA) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin and anti-mycotic agent, as previously described [27].

Adenovirus construct preparation
Adenovirus constructs were generated as previously reported [28]. Briefly, the full-length cDNAs of human IGFBP-3 and -5 were obtained by RT-PCR using total RNA extracted from primary human fibroblasts . The cDNAs were  subcloned into the shuttle vector pAdlox and used for the preparation of  replication-deficient adenovirus serotype 5 expressing IGFBP-3 (Ad3),  IGFBP-

Purification of caveolae-enriched membrane fractions
Caveolae-enriched membrane fractions were purified as previously described [34]. Briefly, human lung fibroblasts were plated on 100 mm dishes and cultured for 48

Western blot analysis
Supernatants, cellular lysates and ECM were obtained from cultured fibroblasts as previously described [28] with some modifications. Briefly, 2.0 ϫ 10 5 primary fibroblasts were cultured in 35 mm wells. Lysates were harvested using RIPA buffer containing protease inhibitor cocktail. Cellular supernatants and lysates were resuspended in 6ϫ SDS gel-loading buffer. ECM was scraped directly in 200 l 2ϫ SDS gel-loading buffer as previously described [28].

Preparation of fibroblast nuclear and cytoplasmic extracts
Nuclear and cytoplasmic fractions were extracted from cultured human and mouse fibroblasts as previously described [36]. Briefly, 1 ϫ 10 6 fibroblasts were cultured in 100 mm dishes and scraped with 400 l of Buffer A [

hr, and incubated with anti-IGFBP-5 (Gropep Ltd.) or anti-Cav-1 (BD transduction Laboratories) antibodies followed by Alexa Fluor ® 488-or 555-conjugated donkey anti-mouse or anti-rabbit antibodies (Invitrogen). Appropriate
IgG was used as a control antibody. Hoechst (Sigma-Aldrich) was used to identify nuclei. Images were taken on an Olympus Fluoview 1000 microscope (Olympus America, Inc., Melville, NY, USA) using identical camera settings.

Statistical analysis
Statistical comparisons were performed using the paired or unpaired Student's t-test as appropriate.

Caveolin-1 translocates to the nucleus in IGFBP-5-expressing fibroblasts
Since IGFBP-5 is a secreted protein that translocates to the nucleus to exert its transcription-regulatory activity [20], we speculated that the interaction of Cav-1 with IGFBP-5 on the cell membrane may result in nuclear trafficking of both proteins. We therefore examined Cav-1 levels in nuclear extracts of IGFBP-5expressing primary fibroblasts. As shown in Figure 2A, the relative distribution of Cav-1 in cytoplasmic and nuclear fractions of nontreated (NT: left panel) and cAd-treated (right panel) primary fibroblasts was comparable. In contrast, Cav-1 levels were 40% greater in nuclear extracts of IGFBP-5-expressing fibroblasts ( Fig. 2A, right part). The nuclear localization of Cav-1 was further confirmed using immunofluorescence. Immunocytostaining of Cav-1 and IGFBP-5 revealed the presence of Cav-1 in the nuclei of IGFBP-5-expressing fibroblasts (Fig. 2B). Since Jurgeit et al. had reported that IGFBP-5 is present in intracellular vesicles outside the nucleus [37], we extended our confirmation of Cav-1 and IGFBP-5 co-localization and nuclear translocation using electron microscopy (Fig. 3). Vesicle-like structures were detected in the cytoplasm and the nucleus of IGFBP-5 expressing fibroblasts (Fig. 3B-F) but were not present in control fibroblasts (Fig. 3A). Extensive analysis of these vesicle-like structures revealed the absence of a membrane, suggesting that they consisted of protein aggregates. Upon entry in these nucleus, aggregates of various sizes seemed to fuse and form larger and denser aggregates (Fig. 3C-F). Using immuno-electron microscopy, we confirmed that both Cav-1 and IGFBP-5 are components of these aggregates in IGFBP-5-expressing fibroblasts (Fig. 3G-L).

IGFBP-5 compartmentalization is altered in cytoplasmic and nuclear extracts purified from caveolin-1 deficient fibroblasts
We next sought to determine whether Cav-1 is required for IGFBP-5 nuclear translocation. Levels of IGFBP-5 were compared in cytoplasmic and nuclear extracts from IGFBP-5-expressing WT and Cav-1 -/fibroblasts. As shown in Figure 4C and F, compared to WT fibroblasts, Cav-1 -/fibroblasts had significantly lower levels of IGFBP-5 in both the nucleus and cytoplasm. Similar results were observed for IGFBP-3 in Cav-1 -/fibroblasts infected with Ad3 ( Fig. 4D and F). To exclude the possibility that Cav-1 deficiency prevents efficient adenoviral infection, we infected the fibroblasts with an adenovirus expressing human Egr-1, a transcription factor known to localize to the nucleus [38]. Egr-1 levels were comparable in nuclear extracts from WT and Cav-1 -/fibroblasts ( Fig. 4E and F), suggesting that reduced IGFBP nuclear translocation in Cav-1 null fibroblasts is not due to hindrance of adenoviral infection and that nuclear compartmentalization of IGFBP-3 and IGFBP-5 requires Cav-1.

Absence of caveolin-1 is associated with low intracellular and high extracellular levels of IGFBP-5
Having observed a lack of IGFBP-5 in the cytoplasmic and nuclear fractions of IGFBP-5-expressing Cav-1 -/fibroblasts, we compared intracellular IGFBP-5 levels to those secreted and deposited  Figure 5A, IGFBP-5 levels were significantly reduced in lysates of Cav-1 -/fibroblasts. This was further confirmed using immunocytochemistry (Fig. 5B). In contrast to cellular lysates, IGFBP-5 levels in the extracellular milieu in both media conditioned by IGFBP-5 expressing Cav-1 -/fibroblasts and the ECM deposited by the cells were higher than those in their WT counterparts (Fig. 5C). IGFBP-5 can be proteolytically cleaved by several different proteases, and increased extracellular levels of IGFBP-5 can result from reduced IGFBP-5 degradation. To compare IGFBP-5 degradation in WT and Cav-1 -/fibroblasts, we used a degradation assay. As shown in Figure 5D, levels of intact rBP5 were comparable in the presence of media conditioned by WT and Cav-1 -/-, indicating that the absence of Cav-1 does not modulate the extracellular degradation of IGFBP-5. To further evaluate the effects of Cav-1 on IGFBP-5 expression, we examined steady-state mRNA levels of IGFBP-5 in WT and Cav-1 -/fibroblasts. At baseline, Cav-1 -/lung fibroblasts had significantly higher IGFBP-5 mRNA levels compared to WT cells (Fig. 5E), likely a compensatory response to the reduced intracellular protein levels of IGFBP-5 in Cav-1 -/fibroblasts.  Figure 6A,  (Fig. 6B, C). The decrease in IGFBP-5 uptake reached significance at 30 min. (Fig. 6B and C, right panels).

IGFBP-5 trafficking can be restored by restitution of Cav-1 function
The Cav-1 CSD peptide can bind to a variety of proteins, and restoration of Cav-1 function can be accomplished by delivery of Cav-1 CSD [39]. We used a cell-permeable Cav-1 CSD peptide at concentrations shown by others to restore Cav-1 function [12,16], to evaluate whether restoration of Cav-1 function facilitates normal uptake of IGFBP-5. Briefly, Cav-1 -/fibroblasts were treated with 5 M Cav-1 CSD peptide for 1 hr, and then purified FLAG-tagged IGFBP-5 was added. Fibroblasts were harvested and IGFBP-5 levels were detected by immunoblotting. As shown in Figure 7, restoration of Cav-1 function in Cav-1 -/fibroblasts using Cav-1 CSD peptide dramatically increased IGFBP-5 internalization, thus restoring IGFBP-5 trafficking.

IGFBP-5 protects ECM components from degradation
We have previously demonstrated increased expression, secretion, and ECM deposition of IGFBP-5 in primary fibroblasts from patients with SSc and IPF [27,28]. We have also reported that IGFBP-5 induces production of collagen and FN and binds these ECM components [28]. In addition, we and others have reported reduced levels of Cav-1 in primary fibroblasts from patients with SSc and IPF [12,13]. In view of these findings, we speculated that increased IGFBP-5 deposition in the ECM of fibrotic cells resulted from impairment of IGFBP-5 cellular entry due to low expression     confirming our previous observations [28] and establishing that IGFBP-5 can bind to FN and afford it protection from proteolytic activity. In the presence of conditioned media supplemented with PMSF and protease inhibitor cocktail-, but not EDTA-treated supernatants, FN levels in the ECM were higher, suggesting that FN was protected from degradation. Although degraded FN fragments could not be detected using the FN antibody, taken together, our results indicate that IGFBP-5-bound FN in ECM is protected from proteolytic degradation. In primary pulmonary fibroblasts, IGFBP-5 co-localized with Cav-1 and both proteins formed complexes. In addition, Cav-1 CSD peptide significantly increased the intracellular uptake of IGFBP-5, suggesting an interaction between the CSD peptide and IGFBP-5. In other words, IGFBP-5 might be a direct target of Cav-1 or could be an indirect target as a Cav-1-bound protein. In this regard, the consensus sequences of CSD peptide ligands have been identified [40]. The sequences, XXXXX, XXXXXX and XXXXXXX, where is any of the aromatic acids (F, W or Y) and X is any amino acid, are the first described as Cav-1 binding domains responsible for the interaction of Cav-1 with other proteins. Signalling molecules, G-protein receptors and growth factor receptors, including mitogen-activated protein (MAP) kinases, eNOS, endothelin R, epidermal growth factor (EGF)-R and IGFBP-3 that are known to be caveolin-associated proteins, contain these motifs [40,41]. Broader consensus Cav-1 binding sequences, in which a hydrophobic amino acid (I, V or L) replaces aromatic acids in addition to F, W or Y, were subsequently reported [42]. We speculate that IGFBP-5 might be a direct target of Cav-1 since IGFBP-5 contains the sequence ICWCVDKY 247 , an imperfect consensus sequence with homology to XZXXXX or ZXXXXXXZ, where Z stands for F, W, Y, I, V or L. [20,24,25]. Since inhibition of protein secretion abolished IGFBP nuclear localization, it was deduced that nuclear IGFBP-3 and -5 are derived from the secreted proteins [20,41]. We have also reported that nuclear translocation of IGFBP-5 occurs in a time-dependent, MAPK-dependent and IGF-I independent manner [30]. The internalization and nuclear transport of IGFBP-3, a protein with significant homology to IGFBP-5 containing a nuclear localization sequence, is documented in a variety of cells and includes caveolin-and clathrin-dependent pathways [24,25,41]. However, pathways mediating the internalization of IGFBP-5 have not been reported, especially in primary cells such as fibroblasts. Our current study identified the caveolin pathway as a mediator of IGFBP-5 internalization in primary fibroblasts. It is not likely that the caveolindependent pathway is the sole mediator of IGFBP-5 uptake, since neither caveolar disruption nor absence of Cav-1 completely inhibited IGFBP-5 uptake. As both caveolin-and transferrin-mediated endocytosis are responsible for IGFBP-3 internalization [41], other pathways, including the transferrin-dependent classical pathway [43] and heparan sulphate proteoglycan-associated internalization [44] may also be involved in IGFBP-5 endocytosis. The latter is likely based on the ability of IGFBP-5 to interact with heparan sulphate proteoglycans [45].

Nuclear translocation of IGFBP-3 and IGFBP-5 has been reported in vascular smooth muscle cells, T47D human breast carcinoma cells and Chinese hamster ovary cells
Electron microscopy revealed the presence of cytoplasmic and nuclear aggregates composed mainly of IGFBP-5 in IGFBP-5expressing fibroblasts. Interestingly, Cav-1 was also detected inside these aggregates, suggesting that they may have originated from the plasma membrane. This suggests a scenario whereby secreted IGFBP-5 was taken up into caveolae, which subsequently formed intracellular vesicle-like structures that trafficked to the nucleus. Our finding is in partial agreement with a recent report showing that IGFBP-5 is found in vesicular structures in T47D cells [37]. However, in this report, IGFBP-5-containing vesicles were not detected in the nucleus. Thus unlike the observations in T47D cells, we clearly show using electron microscopy that IGFBP-5 translocates to the nuclear compartment in inclusions that lack a membrane and are not true vesicles. The fact that IGFBP-5 forms multimers [46] may explain, at least in part, its ability to form aggregates. The differences between our observations and those of Jergeit et al. may be attributed to the cell types as they used immortalized cancer cell lines whereas we used primary fibroblasts, or the use of replication-deficient adenovirus to drive the expression of IGFBP-5 in our system. However the latter is unlikely as expression of IGFBP-3 using the same adenoviral construct did not result in the formation of vesicles or aggregates (data not shown). One of the limitations of immuno-electron microscopy used to confirm the presence of IGFBP-5 and Cav-1 in the nucleus is that detection of proteins is limited to those that are abundant as sections (70 nm) are notably thinner than those use for immunohistochemistry or immunofluorescence staining (6 M). Thus, although few 10 nm gold particles identifying Cav-1were detected, the shear presence of these particles suggests that Cav-1 is abundant in the nuclei of IGFBP-5-expressing fibroblasts.
Although Cav-1 is a major membrane protein, our data demonstrate that Cav-1 is also localized in the nucleus. Several studies support our findings. For example, vascular endothelial growth factor (VEGF) induces nuclear translocation of Flk-1/KDR, eNOS and Cav-1 in vascular endothelial cells [47]. Increased nuclear localization of Cav-1 was also reported in human diploid fibroblasts in H2O2-induced premature senescence [48]. Furthermore, Dittmann et al. reported that Cav-1 dependent EGF-R internalization was induced by irradiation in a src-dependent manner, and subsequently Cav-1 and EGF-R were shuttled into the nucleus and induced DNA damage [49]. Likewise, Cav-1 mediates the internalization and nuclear compartmentalization of several proteins, and likely has a regulatory function. Thus, the role of nuclear Cav-1, in conjunction with the pro-fibrotic activity of IGFBP-5, is novel and warrants further exploration.
Since it has been reported that IGFBP-5 can interact with several ECM components, including FN, collagen and proteoglycans [28,45,50], we performed an ECM degradation assay to explore the effects that reduced Cav-1 levels and resulting increased extracellular IGFBP-5 deposition have on the fibrotic phenotype. Our results show that IGFBP-5 bound FN in the ECM and as a result protected FN from proteolytic degradation in vitro. Recently, decreased Cav-1 expression in skin and lung fibroblasts of patients with SSc and IPF has been reported [12,13]. Furthermore, we have reported increased IGFBP-5 in fibroblasts, skin and lung tissues from patients with these same diseases [27,28]. We now show that Cav-1 deficiency results in increased deposition of secreted IGFBP-5 in the ECM and that this IGFBP-5 can bind ECM components and prevent their degradation. Our findings establish a mechanism by which decreased Cav-1 and increased IGFBP-5 in concert promote the progression and perpetuation of fibrosis. We thus propose a scenario where SSc and IPF fibroblasts secrete IGFBP-5 [27,28]. Secreted IGFBP-5 induces production of collagen and FN [29] and accumulates in the ECM due to inefficient internalization in fibrotic cells that have reduced Cav-1 levels [12,13]. Extracellular IGFBP-5 binds components of the ECM and protects them from proteolytic degradation, thus perpetuating the accumulation of ECM components and supporting the fibrotic phenotype. In addition, we recently reported that IGFBP-5 has a chemoattractant activity and promotes the migration of mononuclear cells into lung tissues [29,30,32] and the transition of fibroblasts and epithelial cells to a myofibroblastic phenotype [29][30][31][32]. Both immune cell infiltration and the activation of fibroblasts into myofibroblasts are implicated in the development of fibrosis. Thus, IGFBP-5 contributes to the fibrotic phenotype via promoting the production and deposition of ECM components, protecting ECM components from degradation, the recruitment of mononuclear cells and fibroblasts, and the activation of fibroblasts.
Restoration of Cav-1 function using Cav-1 CSD peptide has been suggested as a potential therapeutic strategy in SSc and IPF patients [16], as Cav-1 inhibits TGF-␤ signalling by promoting the degradation of the TGF-␤ receptor and preventing Smad 2 phosphorylation [14]. Systemic administration of Cav-1 CSD peptide to bleomycintreated mice dramatically inhibited epithelial cell apoptosis, inflammatory cell infiltration, activation of signalling molecules, and expression of ECM components [16]. In our study, treatment with Cav-1 CSD peptide induced rapid uptake of extracellular IGFBP-5, thus reducing extracellular levels of IGFBP-5. Taken together, these findings suggest that administration of Cav-1 CSD peptide may have beneficial effects in fibrosis. However, in contrast to fibroblasts, an increase of Cav-1 expression in endothelial cells was reported during lung fibrogenesis [10]. In addition, pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension have increased Cav-1 levels which contributes to pulmonary artery smooth muscle cell proliferation and hypertrophy of the pulmonary vascular wall [51], an effect opposite the phenotype described in Cav-1 null mice [17,18]. Thus, as for any potential therapy tested in vitro and in vivo in animals, a therapeutic approach using CSD peptide should be explored cautiously.
In summary, we show that IGFBP-5 is internalized via Cav-1 containing lipid rafts and traffics to the nucleus in vesicular-like inclusions. IGFBP-5 deposition in the ECM is accentuated in Cav-1-deficient lung fibroblasts, promoting the fibrotic phenotype by supporting the accumulation of IGFBP-5 in the extracellular milieu and its binding to components of the ECM, thus preventing their degradation. As decreased expression of Cav-1 in fibroblasts from SSc and IPF patients has been reported [12,13], our findings provide new insights into mechanisms used by IGFBP-5 to induce and promote fibrosis and the role of Cav-1 in the compartmentalization and trafficking of IGFBP-5.