Regulation of insulin-like growth factors I and II and their binding proteins in human bone marrow stromal cells by dexamethasone

Glucocorticoids inhibit the proliferation, but induce the differentiation, of bone marrow stromal cells into osteoblast-like cells. The mechanisms, however, are still conjectural. Since insulin-like growth factors (IGFs) have profound effects on osteoblast growth and differentiation, it is possible that glucocorticoids exert their effects on bone marrow stromal cells in part via regulation of IGFs. Therefore, we analyzed the effects of dexamethasone (Dex) on the expression of IGF I and IGF II in cultured preosteoblastic normal human bone marrow stromal cells (HBMSC). Whereas Dex decreased the concentration of IGF I in the conditioned medium since early in the treatment, the concentration of IGF II was increased progressively as culture period lengthened. As the activities of IGF I and IGF II are regulated by the IGF binding proteins (IGFBPs), we analyzed the effects of Dex on the expression of IGFBPs. Dex increased IGFBP-2 in a time-dependent manner. The increase in IGFBP-2, however, was only to the same extent as that of IGF II at most, depending on the length of treatment. Therefore, the increase in IGFBP-2 would dampen, but not eliminate, the increased IGF II activities. By contrast, Dex decreased IGFBP-3 levels, the latter increasing the bioavailability of IGF II. Although IGFBP-4 mRNA levels were stimulated by Dex, IGFBP-4 concentration in the conditioned medium was unchanged as measured by RIA. IGFBP-5 and IGFBP-6 mRNA levels were decreased by Dex in a time-dependent fashion. IGFBP-5 protein level was also decreased 1–4 days after Dex treatment. IGFBP-1 mRNA was not detectable in HBMSC. These accumulated data indicate that Dex regulates IGF I and IGF II and their binding proteins differentially in normal human bone marrow stromal cells. The progressive increase in IGF II may contribute to Dex-induced cell differentiation. J. Cell. Biochem. 71:449–458, 1998. © 1998 Wiley-Liss, Inc.     

IGF binding proteins and their functions

The insulin-like growth factor (IGF) binding proteins (IGFBPs) have several functions, including transporting the IGFs in the circulation, mediating IGF transport out of the vascular compartment, localizing the IGFs to specific cell types, and modulating both IGF binding to receptors and growth-promoting actions. The functions of IGFBPs appear to be altered by posttranslational modifications. IGFBP-3, -4, -5, and -6 have been shown to be glycosylated. Likewise all the IGFBPs have a complex disulfide bond structure that is required for maintenance of normal IGF binding. IGFBP-2, -3, -4, and -5 are proteolytically cleaved, and specific proteases have been characterized for IGFBP-3, -4, and -5. Interestingly, attachment of IGF-I or II to IGFBP-4 results in enhancement of proteolysis, whereas attachment of either growth factor to IGFBP-5 results in inhibition of proteolytic cleavage. Cleavage of IGFBP-3 results in the appearance of a 31 kDa fragment that is 50-fold reduced in its affinity for the IGF-I or IGF-II. In spite of the reduction in its affinity, this fragment is capable of potentiating the effect of IGF-I on cell growth responses; therefore, proteolysis may be a specific mechanism that alters IGFBP modulation of IGF actions. Other processes that result in a reduction in IGF binding protein affinity are associated with potentiation of cellular responses to IGF-I and -II. Specifically, the binding of IGFBP-3 to cell surfaces is associated with its ability to enhance IGF action and with a ten- to 12-fold reduction in its affinity for IGF-I and IGF-II. Likewise, binding of IGFBP-5 to extracellular matrix (ECM) results in an eightfold reduction in its affinity and a 60% increase in cell growth in response to IGF-I. Another post-translational modification that modifies IGFBP activity is phosphorylation. IGFBP-1, -2, -3, and -5 have been shown to be phosphorylated. Phosphorylation of IGFBP-1 results in a sixfold enhancement in its affinity for IGF-I and -II. Following this enhancement of IGFBP-1 affinity, this binding protein loses its capacity to potentiate IGF-I growth-promoting activity. Future studies using site-directed mutagenesis to modify these proteins should enable us to determine the effect of these posttranslational modifications on the ability of IGFBPs to modulate IGF biologic activity. © 1993 Wiley-Liss, Inc.     

Tetrodotoxin attenuates isoproterenol-induced hypertrophy in H9c2 rat cardiac myocytes

Thyroid stimulating hormone (TSH) is shown to have definite anabolic effects on skeletal metabolism. Previous studies have demonstrated that Insulin-like growth factors (IGF-I and IGF-II) and their six high affinity binding proteins (IGFBPs 1-6) regulate proliferation and differentiation of bone-forming osteoblasts. The current study was intended to determine whether the anabolic effects of TSH on human osteoblastic (SaOS2) cells are mediated through insulin-like growth factor system components. TSH given at 0.01 ng to 10 ng/ml dose levels for 24 and 48 h significantly increased human osteoblastic (SaOS2) cell proliferation and alkaline phosphatase activity, the differentiation marker. TSH significantly increased IGFs (IGF-I and IGF-II) mRNA expression after 6 and 24 h and their protein levels after 24 and 48 h of treatment, respectively. Unlike the IGFs, the IGFBPs responded differently to TSH treatment. Though there were some inconsistencies in the regulation of stimulatory IGF binding protein-3 and -5 by TSH treatment, there was an overall increase at the mRNA abundance and protein levels. Again, the inconsistency persisted at the regulation of the inhibitory IGFBPs 2, 4, and 6 especially at the level of mRNA expression due to TSH treatment, there is an overall decrease in the levels of IGFBP-2, 4, and 6 in the conditioned media (CM) of SaOS2 cell cultures. The IGFBP proteases which control the availability of IGFs are also regulated by hormones. Pregnancy-Associated Plasma Protein-A (PAPP-A) is responsible for the proteolysis of IGFBP-4. TSH treatment significantly unregulated the expression of PAPP-A both at mRNA and protein levels. In conclusion, TSH promotes human osteoblastic (SaOS2) cell proliferation and differentiation by upregulating IGFs and their stimulatory IGF binding proteins and down regulating the inhibitory IGF binding proteins.     

An in situ hybridization study of the insulin-like growth factor system in developing condylar cartilage of the fetal mouse mandible

The objective of this study was to investigate the involvement of the insulin-like growth factor (IGF) system in the developing mandibular condylar cartilage and temporomandibular joint (TMJ). Fetal mice at embryonic day (E) 13.0-18.5 were used for in situ hybridization studies using [35S]-labeled RNA probes for IGF-I, IGF-II, IGF-I receptor (-IR), and IGF binding proteins (-BPs). At E13.0, IGF-I and IGF-II mRNA were expressed in the mesenchyme around the mandibular bone, but IGF-IR mRNA was not expressed within the bone. At E14.0, IGF-I and IGF-II mRNA were expressed in the outer layer of the condylar anlage, and IGF-IR mRNA was first detected within the condylar anlage, suggesting that the presence of IGF-IR mRNA in an IGF-rich environment triggers the initial formation of the condylar cartilage. IGFBP-4 mRNA was expressed in the anlagen of the articular disc and lower joint cavity from E15.0 to 18.5. When the upper joint cavity was formed at E18.5, IGFBP-4 mRNA expression was reduced in the fibrous mesenchymal tissue facing the upper joint cavity. Enhanced IGFBP-2 mRNA expression was first recognized in the anlagen of both the articular disc and lower joint cavity at E16.0 and continued expression in these tissues as well as in the fibrous mesenchymal tissue facing the upper joint cavity was observed at E18.5. IGFBP-5 mRNA was continuously expressed in the outer layer of the perichondrium/fibrous cell layer in the developing mandibular condyle. These findings suggest that the IGF system is involved in the formation of the condylar cartilage as well as in the TMJ.     

Insulin-like growth factor binding proteins localize to discrete cell culture compartments in periosteal and osteoblast cultures from fetal rat bone

Insulin-like growth factor (IGF)-I and IGF-II are expressed at biologically effective levels by bone cells. Their stability and activity are modulated by coexpression of IGF binding proteins (IGFBPs). Secreted IGFBPs may partition to soluble, cell-associated, and matrix-bound compartments. Extracellular localization may sequester, store, or present IGFs to appropriate receptors. Of the six IGFBPs known, rat osteoblasts synthesize all but IGFBP-1. Of these, IGFBP-3, -4, and -5 mRNAs are induced by an increase in cAMP. Little is known about extracellular IGFBP localization in bone and nothing about IGFBP expression by nonosteoblastic periosteal bone cells. We compared basal IGFBP expression in periosteal and osteoblast bone cell cultures and assessed the effects of changes in cAMP-dependent protein kinase A or protein kinase C. Basal IGFBP gene expression differed principally in that more IGFBP-2 and -5 occurred in osteoblast cultures, and more IGFBP-3 and -6 occurred in periosteal cultures. An increase in cAMP enhanced IGFBP-3, -4, and -5 mRNA and accordingly increased soluble IGFBP-3, -4, and -5 and matrix-bound IGFBP-3 and -5 in both bone cell populations. In contrast, protein kinase C activators suppressed IGFBP-5 mRNA, and its basal protein levels remained very low. We also detected low M_r bands reactive with antisera to IGFBP-2, -3, and -5, suggesting proteolytic processing or degradation. Our studies reveal that various bone cell populations secrete and bind IGFBPs in selective ways. Importantly, inhibitory IGFBP-4 does not significantly accumulate in cell-associated compartments, even though its secretion is enhanced by cAMP. Because IGFBPs bind IGFs less tightly in cell-bound compartments, they may prolong anabolic effects by agents that increase bone cell cAMP. J. Cell. Biochem. 71:351–362, 1998. © 1998 Wiley-Liss, Inc.     

Mitogenic and antiapoptotic effects of insulin-like growth factor binding protein-6 in the human osteoblastic osteosarcoma cell line Saos-2/B-10.

Insulin-like growth factor (IGF) I is a potent mitogen for human osteosarcoma cells such as the Saos-2/B-10 cell line. IGF binding proteins (IGFBPs) prevent stimulation of DNA synthesis by IGFs. In contrast to recombinant human (rh) IGFBP-2, -3, -4, and -5, 10-100 nM rhIGFBP-6 stimulated [(3)H]thymidine incorporation into DNA and multiplication of Saos-2/B-10 cells. Upon withdrawal of serum, 30 nM IGFBP-6 also decreased apoptosis (within 4 h) and increased protein content and sodium-dependent phosphate uptake (within 24 h), but less potently than IGF I. (125)I-labeled rhIGFBP-6 did not bind to the cells, and cold IGFBP-6 did not affect (125)I-labeled IGF I binding. Production of IGF I, IGF II, and IGFBP-6 by the cells or significant degradation of rhIGFBP-6 could not be detected within 24 h of incubation. Thus, among the rhIGFBPs tested, rhIGFBP-6 is unique in stimulating osteosarcoma cell growth. Furthermore, it has an antiapoptotic effect.     

Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins.

The insulin-like growth factor-binding proteins (IGFBPs) comprise a family of six related peptides that interact with high affinity with IGFs. IGFBPs compete with IGF receptors for IGF binding, and as a consequence of this competition they can affect cell growth. In addition, IGF-independent regulatory mechanisms of IGFBPs have been described. Despite their common property to interact with IGFs every IGFBP is expressed in a tightly regulated time- and tissue-specific manner suggesting that each protein may have its own distinct functions. Several transgenic mouse models overexpressing IGFBP-1, -2, -3, or -4 were developed in the past few years. Brain abnormalities were a common feature of IGFBP-1 transgenic models. Individual strains showed alterations in glucose homeostasis, reproductive performance, and a reduction of somatic growth as the most prominent phenotypes. The latter was also the main effect observed in IGFBP-2 transgenic mice. The overexpression of IGFBP-3 under the control of an ubiquitous promoter resulted in selective organomegaly, whereas mammary gland-targeted expression of this protein caused an altered involution after pregnancy in this organ. Tissue-specific overexpression of IGFBP-4 resulted in hypoplasia and reduced weight of smooth muscle-rich tissues such as bladder, aorta, and stomach. This review summarizes the current knowledge about the actions of IGFBPs in vivo based on the presently established transgenic mice.     

Insulin-like growth factor binding proteins (IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3.

Insulin-like growth factors (IGFs) are important growth regulators of both normal and malignant prostate cells. Their action is regulated by six insulin-like growth factor binding proteins (IGFBPs). The proteolytic cleavage of IGFBPs by various proteases decreases dramatically their affinity for their ligands and therefore enhances the bioavailability of IGFs. To elucidate the putative biological role of prostatic kallikreins hK2 and hK3 (prostate-specific antigen) in tumour progression, we analyzed the degradation of IGFBP-2, -3, -4 and -5 by these two tissue kallikreins. We found that hK3, already characterized as an IGFBP-3 degrading protease, cleaved IGFBP-4 but not IGFBP-2 and -5, whereas hK2 cleaved all of the IGFBPs much more effectively, and at concentrations far lower than those reported for other IGFBP-degrading proteases. The proteolytic patterns after cleavage of IGFBPs by hK2 and hK3 were similar and were not modified in the presence of IGF-I. Heparin, but not other glycosaminoglycans, enhanced dramatically the ability of hK3 but not hK2 to degrade IGFBP-3 and IGFBP-4. More importantly, the IGFBP fragments generated by hK2 and hK3 had no IGF-binding capacity, as assessed by Western ligand blotting. Our results suggest that the prostatic kallikreins hK2 and hK3 may influence specifically the tumoral growth of prostate cells through the degradation of IGFBPs, to increase IGF bioavailability.     

Identification of insulin-like growth factor binding proteins from cultured human epidermal keratinocytes

The role and mechanisms of action of insulin-like growth factors (IGFs) in skin remain unclear. Epidermal keratinocytes possess IGF-I receptors and are responsive to IGF-I, which is primarily derived from underlying dermal fibroblasts. IGF binding proteins (IGFBPs), also synthesized by fibroblasts, may be involved in paracrine targeting of IGF-I to its receptors. We therefore examined whether human keratinocytes synthesize IGFBPs and their mRNAs. Following culture in complete medium (containing bovine pituitary extract and epidermal growth factor) Western ligand blotting (WLB) of cell conditioned medium revealed a major band of 32 kD, a less abundant IGFBP of 24 kD at all passages, and a 37–42 kD IGFBP which increased in abundance in late passage. Immunoprecipitation followed by WLB confirmed that the predominant 32 kD band was IGFBP-2. Radioimmunoassay of IGFBP-1, -3, and -6 revealed detectable levels of IGFBP-3 and significant levels of IGFBP-6, but not IGFBP-1. Northern analysis following culture in complete medium revealed that at early passage IGFBP-1, -2, -4, and -6 mRNAs were detectable. IGFBP-3 and -5 mRNAs were not detectable. Following culture in growth factor-free medium a 37–42 kD band, consistent with IGFBP-3, was predominant and a 24 kD band consistent with IGFBP-4 was also present. These data demonstrate the expression of a distinct pattern of IGFBPs by cultured human keratinocytes dependent on culture conditions. Keratinocyte-derived IGFBPs are likely to play a role in the transport and targeting of IGF-I from dermally derived fibroblasts to the epidermis. © 1995 Wiley-Liss, Inc.     

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.     

Regulation of renal growth and IGFBP-4 expression by triiodothyronine during rat development.

The insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs), which regulate IGF activity, play a fundamental role in renal cell proliferation and differentiation. The thyroid hormone is considered to be required for kidney development; excess induces local hypertrophy and hyperplasia. The aim of the present study was to investigate the possible involvement of the IGF/IGFBP system in thyroid hormone-induced renal growth during the development of the rat. Our results show that thyroid hormone withdrawal by 6-propyl-2-thiouracil (PTU)-treatment of rats at all ages had no effect on renal IGFBP-4 mRNA levels, whereas the abundance of the serum protein was decreased compared to controls. Intraperitoneal triiodothyronine (T3) administration to hypothyroid rats resulted in renal hypertrophy associated with a significant upregulation of IGFBP-4 expression with increased levels of renal IGFBP-4 mRNA and serum protein. T3-induced upregulation of IGFBP-4 expression suggests the involvement of the local IGF/IGFBP system in T3-induced renal hypertrophy.     

Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status

The insulin-like growth factors (IGFs) have been implicated in the growth regulation of human breast cancer. Since the IGFs are associated with specific binding proteins (IGFBPs) which may modulate receptor/ligand interactions, production of IGFBPs by breast cancer cells could alter their IGF-dependent growth. This study examined the expression of IGFBPs 4, 5, and 6 in eight breast cancer cell lines (BCCLs) using ribonuclease (RNase) protection assays. IGFBP-4 mRNA was detected in all BCCLs studied. IGFBP-5 expression was higher in estrogen receptor (ER) positive cells, while IGFBP-6 mRNA was detected in only two ER negative BCCLs. We also found that E₂ treatment enhanced the expression of IGFBPs 2, 4, and 5 in T47-D cells. We next studied IGFBP mRNA expression in 40 primary breast tumors. All tumors expressed mRNA for IGFBPs 2–6 but none expressed IGFBP-1 message. IGFBP-3 expression was higher in ER negative tumors, while that of IGFBP-4 and -5 was higher in ER positive specimens. These differences were statistically significant (P < .05). Ligand blot analysis of tumor extracts confirmed the presence of IGFBPs in breast cancer tissues. Thus, differential IGFBP expression in ER positive and negative tumors suggests an important role for this protein in breast cancer biology.     

Insulin-like growth factor binding protein (IGFBP)-3-bound IGF-I and IGFBP-3-bound IGF-II in growth hormone deficiency

In blood, circulating IGFs are bound to six high-affinity IGFBPs, which modulate IGF delivery to target cells. Serum IGFs and IGFBP-3, the main carrier of IGFs, are upregulated by GH. The functional role of serum IGFBP-3-bound IGFs is not well understood, but they constitute the main reservoir of IGFs in the circulation. We have used an equation derived from the law of mass action to estimate serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II, as well as serum free IGF-I and free IGF-II, in 129 control children and adolescents (48 girls and 81 boys) and in 13 patients with GHD. Levels of serum total IGF-I, total IGF-II, IGFBP-1, IGFBP-2 and IGFBP-3 were determined experimentally, while those of IGFBP-4, IGFBP-5 and IGFPB-6, as well as the 12 affinity constants of association of the two IGFs with the six IGFBPs, were taken from published values. A correction for in vivo proteolysis of serum IGFBP-3 was also considered. In controls, serum total IGF-I, total IGF-II, IGFBP-3, IGFBP-3-bound IGF-I, IGFBP-3-bound IGF-II and free IGF-I increased linearly with age, from less than 1 to 15 years, in the two sexes. The concentrations of serum free IGF-I and free IGF-II were approximately two orders of magnitude below published values, as well as below the affinity constant of association of IGF-I with the type-1 IGF receptor. Therefore, it is unlikely that these levels can interact with the receptor. In the 13 patients with GHD, mean (+/- SD) SDS of serum IGFBP-3-bound IGF-I was -2.89 +/- 0.97. It was significantly lower than serum total IGF-I, free IGF-I or IGFBP-3 SDSs (-2.35 +/- 0.83, -1.12 +/- 0.78 and -2.55 +/- 1.07, respectively, p = 0.0001). The mean SDS of serum total IGF-II, IGFBP-3-bound IGF-II and free IGF-II were -1.25 +/- 0.68, -2.03 +/- 0.87 and 0.59 +/- 1.10, respectively, in GHD. In control subjects, 89.8 +/- 4.47% of serum total IGF-I and 77.3 +/- 9.4% of serum total IGF-II were bound to serum IGFBP-3. In patients with GHD, the mean serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II were 8.63 +/- 8. 53 and 19.1 +/- 14.7% below the respective means of control subjects (p < 0.02). In conclusion, in GHD there was a relative change in the distribution of serum IGFs among IGFBPs, due to the combined effects of the decrease in both total IGF-I and IGFBP-3. As a result, serum IGFBP-3-bound IGF-I and IGFBP-3 bound IGF-II, the main reservoirs of serum IGFs, were severely affected. This suggests that the decrease in serum IGFPB-3-bound IGF-I and IGFBP-3-bound IGF-II might have a negative effect for growth promotion and other biological effects of IGF-I and IGF-II. Finally, the estimation of serum IGFBP-3-bound IGF-I, or the percentage of total IGF-I and IGF-II bound to IGFBP-3, might be useful markers in the diagnosis of GHD.     

The N-terminal subdomain of insulin-like growth factor (IGF) binding protein 6. Structure and interaction with IGFs

Insulin-like growth factor binding proteins (IGFBPs) modulate the activity and distribution of insulin-like growth factors (IGFs). IGFBP-6 differs from other IGFBPs in being a relatively specific inhibitor of IGF-II actions. Another distinctive feature of IGFBP-6 is its unique N-terminal disulfide linkages; the N-domains of IGFBPs 1-5 contain six disulfides and share a conserved GCGCC motif, but IGFBP-6 lacks the two adjacent cysteines in this motif, so its first three N-terminal disulfide linkages differ from those of the other IGFBPs. The contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties and their structure-function relationships have been characterized in part, but the structure and function of the distinctive N-terminal subdomain of IGFBP-6 are unknown. Here we report the solution structure of a polypeptide corresponding to residues 1-45 of the N-terminal subdomain of IGFBP-6 (NN-BP-6). The extended structure of the N-terminal subdomain of IGFBP-6 is very different from that of the short two-stranded beta-sheet of the N-terminal subdomain of IGFBP-4 and, by implication, the other IGFBPs. NN-BP-6 contains a potential cation-binding motif; lanthanide ion binding was observed, but no significant interaction was found with physiologically relevant metal ions like calcium or magnesium. However, this subdomain of IGFBP-6 has a higher affinity for IGF-II than IGF-I, suggesting that it may contribute to the marked IGF-II binding preference of IGFBP-6. The extended structure and flexibility of this subdomain of IGFBP-6 could play a role in enhancing the rate of ligand association and thereby be significant in IGF recognition.     

Differential regulation of insulin-like growth factor binding protein (IGFBP)-2 mRNA in liver and bone cells by insulin and retinoic acid in vitro.

Isolated cells produce insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). Two distinct cell types were studied with regard to IGFBP-2 expression: (i) rat hepatocytes, which produce IGF I at a high rate and thus regulate its plasma concentration; and (ii) rat osteoblasts, which are targets of IGF I action. IGFBP-2 expression is low in hepatocytes prepared from normal adult rats and high in calvaria cells from newborn rats. Retinoic acid stimulates IGFBP-2 production by liver cells. Insulin suppresses both basal and retinoic acid-induced IGFBP-2 mRNA expression in hepatocytes and has no such effect on osteoblasts. Retinoic acid and insulin regulate IGFBP-2 expression in a tissue-specific manner.