Analysis of 17ß-estradiol (E2) role in the regulation of corpus luteum function in pregnant rats: Involvement of IGFBP5 in the E2-mediated actions.

BACKGROUND: In several species, considerably higher levels of estradiol-17 (E2) are synthesized in the CL. E2 has been suggested to participate in the regulation of luteal steroidogenesis and luteal cell morphology. In pregnant rats, several experiments have been carried out to examine the effects of inhibition of luteal E2 synthesis on CL structure and function. METHODS: During days 12-15 of pregnancy in rats, luteal E2 was inhibited by way of daily oral administration of anastrozole (AI), a selective non-steroidal aromatase inhibitor, and experiments were also performed with E2 replacement i.e. AI+ E2 treatments. Luteal tissues from different treatment groups were subjected to microarray analysis and the differentially expressed genes in E2 treated group were further examined for expression of specific E2 responsive genes. Additional experiments were carried out employing recombinant growth hormone preparation and flutamide, an androgen receptor antagonist, to further address the specificity of E2 effects on the luteal tissue. RESULTS: Microarray analysis of CL collected on day 16 of pregnancy post AI and AI+E2 treatments showed significantly lowered cyp19a1 expression, E2 levels and differential expression of a number of genes, and several of them were reversed in E2 replacement studies. From the differentially expressed genes, a number of E2 responsive genes were identified. In CL of AI pregnant rats, non-significant increase in expression of igf1, significant increase in igbp5, igf1r and decrease in expression of Erα were observed. In liver of AI treated rats, igf1 expression did not increase, but GH treatment significantly increased expression that was further increased with AI treatment. In CL of GH and AI+GH treated rats, expression of igfbp5 was higher. Administration of flutamide during days 12-15 of pregnancy resulted in non-significant increase in igfbp5 expression, however, combination of flutamide+AI treatments caused increased protein expression. Expression of few of the molecules in PI3K/Akt kinase pathway in different treatments was determined. CONCLUSIONS: The results suggest a role for E2 in the regulation of luteal steroidogenesis, morphology and proliferation. igfbp5 was identified as one the E2 responsive genes with important role in the mediation of E2 actions such as E2-induced phosphorylation of PI3K/Akt kinase pathway.

Insulin-Like Growth Factor Binding Proteins--an Update.

The insulin-like growth factor (IGF) system is essential for normal growth and development, and its perturbation is implicated in a number of diseases. IGF activity is finely regulated by a family of six high-affinity IGF binding proteins (IGFBPs). 1GFBPs usually inhibit IGF actions but may enhance them under certain conditions. Additionally, IGFBPs bind non-IGF ligands in the extracellular space, cell membrane, cytoplasm and nucleus, thereby modulating cell proliferation, survival and migration in an IGF-independent manner. IGFBP activity is regulated by transcriptional mechanisms as well as by post-translational modifications and proteolysis. Understanding the balance between the various actions of IGFBPs in vivo may lead to novel insights into disease processes and possible IGFBP-based therapeutics.

Exogenous GLP-2 and IGF-I induce a differential intestinal response in IGF binding protein-3 and -5 double knockout mice.

Glucagon-like peptide-2 (GLP-2) action is dependent on intestinal expression of IGF-I, and IGF-I action is modulated by IGF binding proteins (IGFBP). Our objective was to evaluate whether the intestinal response to GLP-2 or IGF-I is dependent on expression of IGFBP-3 and -5. Male, adult mice in six treatment groups, three wild-type (WT) and three double IGFBP-3/-5 knockout (KO), received twice daily intraperitoneal injections of GLP-2 (0.5 µg/g body wt), IGF-I (4 µg/g body wt), or PBS (vehicle) for 7 days. IGFBP-3/-5 KO mice showed a phenotype of lower plasma IGF-I concentration, but greater body weight and relative mass of visceral organs, compared with WT mice (P < 0.001). WT mice showed jejunal growth with either IGF-I or GLP-2 treatment. In KO mice, IGF-I did not stimulate jejunal growth, crypt mitosis, sucrase activity, and IGF-I receptor (IGF-IR) expression, suggesting that the intestinotrophic actions of IGF-I are dependent on expression of IGFBP-3 and -5. In KO mice, GLP-2 induced significant increases in jejunal mucosal cellularity, crypt mitosis, villus height, and crypt depth that was associated with increased expression of the ErbB ligand epiregulin and decreased expression of IGF-I and IGF-IR. This suggests that in KO mice, GLP-2 action in jejunal mucosa is independent of the IGF-I system and linked with ErbB ligands. In summary, the intestinotrophic actions of IGF-I, but not GLP-2, in mucosa are dependent on IGFBP-3 and -5. These findings support the role of multiple downstream mediators for the mucosal growth induced by GLP-2.

Functional roles and clinical values of insulin-like growth factor-binding protein-5 in different types of cancers.

Insulin-like growth factor-binding proteins(IGFBPs) are critical regulators of the mitogenic activity of insulin-like growth factors (IGFs). IGFBP5, one of these IGFBPs, has special structural features, including a nuclear transport domain, heparin-binding motif, and IGF/extracellular matrix/acid-labile subunit-binding sites. Furthermore, IGFBP5 has several functional effects on carcinogenesis and even normal cell processes, such as cell growth, death, motility, and tissue remodeling. These biological effects are sometimes related with IGF (IGF-dependent effects) and sometimes not (IGF-independent effects). The functional role of IGFBP5 is most likely determined in a cell-type and tissue-type specific manner but also depends on cell context, especially in terms of the diversity of interacting proteins and the potential for nuclear localization. Clinical findings show that IGFBP5 has the potential to be a useful clinical biomarker for predicting response to therapy and clinical outcome of cancer patients. In this review, we summarize the functional diversity and clinical importance of IGFBP5 in different types of cancers.

Involvement of IGF binding protein 5 in prostaglandin E(2)-induced cellular senescence in human fibroblasts.

Inflammation is an underlying basis for the molecular alterations that link aging and age-related pathological processes. In a previous study, we found that secretory phospholipase A(2) (sPLA(2)) induced cellular senescence in human dermal fibroblasts (HDFs). To further investigate the association of inflammation with cellular senescence, the effects of PGE(2) on cellular senescence in HDFs were investigated, since PGE(2) is the most abundant prostanoid. PGE(2) treatment induces cellular senescence, as determined by a decrease in cell proliferation and an increase in senescence-associated ß-galactosidase staining. Notably, PGE(2) treatment increased the IGFBP5 protein level. While treatment with PGE(2) antagonists repressed PGE(2)-induced cellular senescence, increasing intracellular cAMP accelerated cellular senescence. Down-regulation of IGFBP5 inhibited PGE(2)-induced cellular senescence. Taken together, these results suggest that PGE(2) may play an important role in controlling cellular senescence of HDFs through the regulation of IGFBP5 and therefore may contribute to inflammatory disorders associated with aging.

Association of porcine IGF binding protein-5 gene with meat quality.

IGFBP-5 is a member of the IGF families. Using PCR-SSCP, genotypic and allelic frequencies were analyzed in 18 pig breeds (n = 600). The association between haplotypes and production performance was analyzed in a Jinhua x Pietrain population family (n = 212, total 24 traits). Two SNPs (T199C and G485A) within the gene were analyzed. The breeds had different genotypic and allelic frequencies. Typically, the Chinese native pig breeds carried a higher allele C and G frequency (over 50%) than those of the European pigs, and only Guangdong Large White and wild boar were at Hardy-Weinberg equilibrium. The pigs carrying the CG haplotype had higher hue, loin, and thigh pH1 values than pigs with the TA haplotype, and pigs with the TA haplotype had the lowest loin pH2 value and highest color-a value among the haplotypes. It is proposed that the IGFBP-5 gene is associated with the variation in meat quality, especially in pH value together with other QTLs on chromosome 15.

Insulin-Like Growth Factor Binding Protein-5 in Physiology and Disease.

Insulin-like growth factor (IGF) signaling is regulated by a conserved family of IGF binding proteins (IGFBPs) in vertebrates. Among the six distinct types of IGFBPs, IGFBP-5 is the most highly conserved across species and has the broadest range of biological activities. IGFBP-5 is expressed in diverse cell types, and its expression level is regulated by a variety of signaling pathways in different contexts. IGFBP-5 can exert a range of biological actions including prolonging the half-life of IGFs in the circulation, inhibition of IGF signaling by competing with the IGF-1 receptor for ligand binding, concentrating IGFs in certain cells and tissues, and potentiation of IGF signaling by delivery of IGFs to the IGF-1 receptor. IGFBP-5 also has IGF-independent activities and is even detected in the nucleus. Its broad biological activities make IGFBP-5 an excellent representative for understanding IGFBP functions. Despite its evolutionary conservation and numerous biological activities, knockout of IGFBP-5 in mice produced only a negligible phenotype. Recent research has begun to explain this paradox by demonstrating cell type-specific and physiological/pathological context-dependent roles for IGFBP-5. In this review, we survey and discuss what is currently known about IGFBP-5 in normal physiology and human disease. Based on recent in vivo genetic evidence, we suggest that IGFBP-5 is a multifunctional protein with the ability to act as a molecular switch to conditionally regulate IGF signaling.

IGFBP-5 induces epithelial and fibroblast responses consistent with the fibrotic response.

Fibrosis involves activation of fibroblasts, increased production of collagen and fibronectin and transdifferentiation into contractile myofibroblasts. The process resembles aspects of wound-healing but remains unresolved and can be life-threatening when manifest in the kidneys, lungs and liver, in particular. The causes are largely unknown, but recent suggestions that repetitive micro-injury results in the eventual failure of epithelial cell repair due to replicative senescence are gaining favour. This is consistent with the onset of fibrotic diseases in middle age. Because epithelial injury often involves blood loss, inflammatory responses associated with the fibrotic response have been considered as therapeutic targets. However, this has proved largely unsuccessful and focus is now switching to earlier events in the process. These include EMT (epithelial-mesenchymal transition) and fibroblast activation in the absence of inflammation. TGFbeta1 (transforming growth factor-beta1) induces both EMT and fibroblast activation and is considered to be a major pro-fibrotic factor. Recently, IGFBP-5 [IGF (insulin-like growth factor)-binding protein-5] has also been shown to induce similar effects on TGFbeta1, and is strongly implicated in the process of senescence. It also stimulates migration of peripheral blood mononuclear cells, implicating it in the inflammatory response. In this paper, we examine the evidence for a role of IGFBP-5 in fibrosis and highlight its structural relationship with other matrix proteins and growth factors also implicated in tissue remodelling.

Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5.

Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.

Insulin-like growth factor-binding protein-5 inhibits osteoblast differentiation and skeletal growth by blocking insulin-like growth factor actions.

Signaling through the IGF-I receptor by locally synthesized IGF-I or IGF-II is critical for normal skeletal development and for bone remodeling and repair throughout the lifespan. In most tissues, IGF actions are modulated by IGF-binding proteins (IGFBPs). IGFBP-5 is the most abundant IGFBP in bone, and previous studies have suggested that it may either enhance or inhibit osteoblast differentiation in culture and may facilitate or block bone growth in vivo. To resolve these contradictory observations and discern the mechanisms of action of IGFBP-5 in bone, we studied its effects in differentiating osteoblasts and in primary bone cultures. Purified wild-type (WT) mouse IGFBP-5 or a recombinant adenovirus expressing IGFBP-5WT each prevented osteogenic differentiation induced by the cytokine bone morphogenetic protein (BMP)-2 at its earliest stages without interfering with BMP-mediated signaling, whereas an analog with reduced IGF binding (N domain mutant) was ineffective. When added at later phases of bone cell maturation, IGFBP-5WT but not IGFBP-5N blocked mineralization, prevented longitudinal growth of mouse metatarsal bones in short-term primary culture, and inhibited their endochondral ossification. Because an IGF-I variant (R3IGF-I) with diminished affinity for IGFBPs promoted full osteogenic differentiation in the presence of IGFBP-5WT, our results show that IGFBP-5 interferes with IGF action in osteoblasts and provides a framework for discerning mechanisms of collaboration between signal transduction pathways activated by BMPs and IGFs in bone.

IGFBP5 is a potential regulator of craniofacial skeletogenesis.

Six known proteins bind to the insulin-like growth factor (IGF) with high affinity. Igfbp5 encodes one of these proteins, which regulates the activity of IGF, but also exerts IGF-independent actions. Using in situ hybridization to detect cells expressing Igfbp5 mRNA, we show that Igfbp5 is expressed in a dynamic pattern in the mouse embryonic craniofacial region. At early stages corresponding to the completion of neural crest migration, Igfbp5 mRNA was found predominantly in the epithelia, whereas when the craniofacial mesenchyme has begun its differentiation into skeletal tissue, Igfbp5-expressing cells surrounded the developing cartilages and bones. Embryos transgenically expressing Igfbp5 in restricted areas of the mesenchyme fated to form craniofacial bones revealed decreased ossification and even deletion of head bones areas. Transgenic expression of a mutant Igfbp5, encoding a product with reduced binding affinity for IGF, led to no skeletal abnormalities, suggesting that the observed negative effects on skeletal development rely on a mechanism that depends on binding to IGF.

Insulin-like growth factor-binding protein-5 (IGFBP-5) acts as a tumor suppressor by inhibiting angiogenesis.

Insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the six members of IGFBP family, important for cell growth control, induction of apoptosis and other IGF-stimulated signaling pathways. In this study, we focused on characterizing the specific function of IGFBP-5 as novel antiangiostatic factor. Overexpression of IGFBP-5 suppressed the tube formation as well as the biological functions of angiostatic activity in vivo. This result is due to the reduced expressions of phosphorylated protein kinase B and phosphorylated endothelial NO synthase, which plays important roles in the regulation of angiogenesis when stimulated by vascular endothelial growth factor. Further, IGFBP-5 expression prevented tumor growth and inhibited tumor vascularity in a xenograft model of human ovarian cancer. These results are the first evidence showing that IGFBP-5 plays a role as tumor suppressor by inhibiting angiogenesis.

Insulin-like growth factor binding protein-5 enhances the migration and differentiation of gingival epithelial cells.

BACKGROUND AND OBJECTIVE: The objective was to define the roles of insulin-like growth factor binding protein-5 (IGFBP-5) in gingival epithelial cells (GEC). Human IGFBP-5 is expressed in many cell types and has diverse biological functions. It stimulates the growth of bone cells and is associated with the impedance of gingival fibroblast apoptosis. In gingival epithelium, IGFBP-5 is expressed in the cells of the differentiated stratum spinosum layer. MATERIAL AND METHODS: Recombinant IGFBP-5 protein treatment and knockdown of IGFBP-5 expression using a lentivirus-delivered short hairpin RNA was carried out in human GEC. Proliferation, apoptosis, anoikis, migration, differentiation and gene expression in GEC were analyzed and molecular images were obtained. RESULTS: The IGFBP-5 had no effect on proliferation, but it slightly suppressed apoptosis and anoikis of GEC. It also induced GEC migration and upregulated the expression of involucrin, transglutaminase-1, keratin and focal adhesion kinase. The IGFBP-5 induced migration partly via an insulin-like growth factor-independent mechanism. The knockdown of IGFBP-5 downregulated the expression of involucrin, transglutaminase-1 and focal adhesion kinase. CONCLUSION: Expression of IGFBP-5 in GEC is associated with anti-apoptosis, migration and differentiation of GEC. These phenotypic effects may be associated with focal adhesion kinase and are advantageous for re-epithelization of GEC and the maintenance of gingival health.

Regulation of insulin-like growth factor (IGF) bioactivity by sequential proteolytic cleavage of IGF binding protein-4 and -5.

The biological activity of IGF-I and -II is controlled by six binding proteins (IGFBPs), preventing the IGFs from interacting with the IGF receptor. Proteolytic cleavage of IGFBPs is one mechanism by which IGF can be released to bind the receptor. The IGFBPs are usually studied individually, although the presence of more than one of the IGFBPs in most tissues suggests a cooperative function. Thus, the IGFBPs are part of regulatory networks with proteolytic enzymes in one end and the IGF receptor in the other end. We have established a model system that allows analysis of the dynamics between IGF, IGFBP-4 and -5, the IGF receptor, and the proteolytic enzyme PAPP-A, which specifically cleaves both IGFBP-4 and -5. We demonstrate different mechanisms of IGF release from IGFBP-4 and -5: cooperative binding to IGF is observed for the proteolytic fragments of IGFBP-5, but not fragments of IGFBP-4. Furthermore, we find that PAPP-A-mediated IGF-dependent cleavage of IGFBP-4 is inhibited by IGFBP-5, which sequesters IGF from IGFBP-4, and that cleavage of both IGFBP-4 and -5 is required for the release of bioactive IGF. Finally, we show that cell surface-localized proteolysis of IGFBP-4 represents the final regulatory step of efficient IGF delivery to the receptor. Our data define a regulatory system in which molar ratios between the IGFBPs and IGF and between the different IGFBPs, sequential proteolytic cleavage of the IGFBPs, and surface association of the activating proteinase are key elements in the regulation of IGF receptor stimulation.

Cellular mechanisms in regulating mammary cell turnover during lactation and dry period in dairy cows.

The mechanisms involved in regulating mammary cell turnover during the pregnancy-lactation cycle in dairy cows are unclear. The objective of present experiment was to describe expression of genes encoding proteins known to be involved in pathways regulating mammary cell proliferation, apoptosis, differentiation, cell survival, and tissue remodeling. Mammary gland biopsies were taken 7 times during the pregnancy-lactation cycle of 10 dairy cows, and samples were analyzed by immunohistochemistry and real-time PCR. Cell proliferation was greatest during the dry period and apoptosis was high in early dry period and early lactation. Based on Fas (tumor necrosis factor receptor superfamily member 6), Fas ligand, and caspase-3, caspase-8, and caspase-9 gene expression, no indication was found of a stage-dependent shift between the extrinsic and intrinsic pathways leading to apoptosis. Gene expression of microsomal glutathione S-transferase (mGST) did not vary significantly, whereas B-cell leukemia/lymphoma 2 (Bcl-2) and BCL2-associated X protein (Bax) gene expression was greatest during the dry period and early lactation and coincided with high cell turnover. Gene expression of early response genes c-Fos, c-Jun, and c-Myc correlated to neither rate of cell proliferation nor plasma concentration of insulin-like growth factor (IGF)-I and insulin. Gene expression of nuclear factor of kappa light chain gene enhancer in B-cells (NFkappaB) and NFkappaB inhibitor alpha was greatest in the periparturient period, and NFkappaB gene expression coincided with an anticipated need for cell survival factors. Expression of transforming growth factor beta (TGF-beta) receptor 1 and 2 mRNA was greatest in early lactation, whereas TGF-beta1 did not vary significant during the pregnancy-lactation cycle. Even though our results on the TGF-beta system did not comply with other studies, the gene expression pattern of the TGF-beta receptors indicates a role in regulating apoptosis in early lactation. Signal transducer and activator of transcription 5 (STAT5) gene expression was high in the periparturient period, which suggests a role for STAT5 in regulation of mammary cell proliferation and differentiation in dairy cows. Expression of tissue-plasminogen activator, plasminogen activator inhibitor-1, and IGF binding protein 5 genes was greatest in early lactation, suggesting a role for IGF binding protein 5 in coordinating regulation of apoptosis and tissue remodeling.

Delayed mammary gland involution in mice with mutation of the insulin-like growth factor binding protein 5 gene.

IGFs (IGF-I and IGF-II) are essential for development, and their bioactivities are tightly regulated by six related IGF-binding proteins (IGFBPs). IGFBP-5 is the most highly conserved binding protein and is expressed in several key developmental lineages as well as in multiple adult tissues including the mammary gland. To explore IGFBP-5 actions in vivo, we produced IGFBP-5 knockout (KO) mice. Whole-body growth, selected organ weights, and body composition were essentially normal in IGFBP-5 KO mice, presumably because of substantial compensation by remaining IGFBP family members. The IGFBP-5 KO mice also exhibited normal mammary gland development and were capable of nursing their pups. We then directly evaluated the proposed role of IGFBP-5 in apoptosis and remodeling of mammary gland during involution. We found that the process of involution after forced weaning was delayed in IGFBP-5 KO mice, with both the appearance of apoptotic cells and the reappearance of adipocytes retarded in mutant mice, compared with controls. We also determined the effects of IGFBP-5 deletion on mammary gland development in pubertal females after ovariectomy and stimulation with estradiol/progesterone. In this paradigm, IGFBP-5 KO mammary glands exhibited enhanced alveolar bud formation consistent with enhanced IGF-I action. These results demonstrate that IGFBP-5, although not essential for normal growth, is required for normal mammary gland involution and can regulate mammary gland morphogenesis in response to hormone stimulation.

Evidence that IGF-binding protein-5 functions as a growth factor.

Recent studies support the concept that IGF-binding protein-5 (IGFBP-5) stimulates bone formation, at least in part, via IGF-independent mechanisms. To evaluate this hypothesis further, we evaluated in vitro and in vivo effects of IGFBP-5 on bone formation parameters using the IGF-I knockout (KO) mouse. Treatment of serum-free cultures of osteoblast clones derived from IGF-I KO mice with recombinant human IGFBP-5 increased both proliferation and alkaline phosphatase (ALP) activity in a dose-dependent manner, an effect comparable to that seen with IGF-I. IGF-II levels from media conditioned by osteoblasts derived from IGF-I KO mouse were below those detectable by RIA. To eliminate possible actions of IGF-II, if any was produced by osteoblasts derived from IGF-I knockout mice, the IGFBP-5 effect was studied in the presence of exogenously added IGFBP-4, a potent inhibitor of IGF-II actions in bone cells. Addition of IGFBP-4 blocked IGF-I- but not IGFBP-5-induced cell proliferation in osteoblasts derived from IGF-I knockout mice. Consistent with in vitro results, a single local injection of IGFBP-5 to the outer periosteum of the parietal bone of IGF-I KO mice increased ALP activity and osteocalcin levels of calvarial bone extracts. The magnitudes of IGFBP-5-induced increases in ALP and osteocalcin in parietal bone extracts of IGF-I KO mice were comparable to those seen in C3H mice. In contrast to IGFBP-5, local administration of IGFBP-4 had no significant effect on bone formation in C3H and IGF-I KO mice. These results provide the first direct evidence to our knowledge that IGFBP-5 functions as a growth factor that stimulates its actions in part via an IGF-independent mechanism.

Insulin-like growth factor binding protein-5 in osteogenesis: facilitator or inhibitor?

The insulin-like growth factors (IGFs) play a central role in controlling somatic growth in mammals and exert anabolic effects on most tissues, including bone. IGF action is mediated by the IGF-I receptor and additionally is regulated by six high-affinity IGF binding proteins (IGFBP-1 through IGFBP-6), of which IGFBP-4 and IGFBP-5 are most abundant in bone. The focus of this brief review is on the role of IGFBP-5 in bone biology. IGFBP-5 has been implicated as a pro-osteogenic factor in several studies but conversely has been shown to act as an inhibitor of bone formation, primarily by interfering with IGF actions on osteoblasts. These potentially contradictory effects of IGFBP-5 in bone are further complicated by observations indicating that IGFBP-5 additionally may function in an IGF-independent way, and may have been accentuated by differences in both experimental design and methodology among published studies. Suggestions are made for a more systematic approach to help discern the true roles of IGFBP-5 in bone physiology.

Evidence that IGF binding protein-5 functions as a ligand-independent transcriptional regulator in vascular smooth muscle cells.

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, (125)I- 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.

Osteoblast differentiation of NIH3T3 fibroblasts is associated with changes in the IGF-I/IGFBP expression pattern.

Insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are essential regulators for osteoblast proliferation and differentiation. It has been reported that Dexamethasone (Dex), an active glucocorticoid (GC) analogue, synergizes the stimulatory effect of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on osteoblast differentiation in the mouse fibroblastic cell line NIH3T3. I investigated whether this stimulatory effect is associated with changes in the expression pattern of the IGF/IGFBP system. Quantitative real-time PCR technology was used to quantify the gene expression levels of the IGF-system during osteoblast differentiation and in response to 1,25(OH)(2)D(3) or Dex alone under serum-containing and serum-free culture conditions. Interestingly, NIH3T3 was shown to express high mRNA levels of IGF-I, IGF-II and IGFBP-5, and low levels of both IGFBP-2 and-6. During osteoblast differentiation (days 6-12), IGF-I mRNA was repressed by more than 60%, while the transcript of IGFBP-5 was markedly up-regulated, by more than 50-fold. Similarly, treatment with Dex alone resulted in a dose-and time-dependent increase in the expression of IGFBP-5 and a decrease in IGF-I mRNA. Treatment with 1,25(OH)(2)D(3) alone increased the mRNA levels of IGF-I and IGFBP-6 by around 4-and 7-fold, respectively, in a dose-and time-dependent manner. In conclusion, my data demonstrated that osteoblast differentiation of NIH3T3 is associated with changes in the expression pattern of IGFs/IGFBPs, which are regulated by glucocorticoid in the presence of 1,25(OH)(2)D(3). Modulation of the IGF/IGFBP levels by glucocorticoid might suggest important roles for the IGF-system in mediating the osteoblast differentiation of the NIH3T3 cell line.