Short chain fatty acid and glucose metabolism in isolated pig colonocytes: modulation by NH4 +

Using monolayer cultures of costal chondrocytes established from four week old Clun Forest lambs, we have demonstrated that, under serum free conditions the cells release three IGFBPs (32, 29 and 21 kDa) into the medium. The most abundant of these—the 32 kDa BP-was shown to be IGFBP-2 by Western blotting. Furthermore we demonstrate that the levels of IGFBP 2 in conditioned medium are acutely increased (6, 12 and 24 h time points) following treatment of cells with bovine GH (1–100 ng/ml).In a parallel set of experiments, using ovine fibroblasts (derived from dermis) we show that IGFBPs of Mr 32, 29 and 21 kDa are also secreted by this cell type. However the relative abundance of these BPs differed from that seen in the chondrocyte cultures, with the 21 kDa species now the most abundant. In addition, prolonged exposure of autoradiographs indicated that fibroblasts secreted a higher Mr IGFBP (most probably ovine BP-3) that was not detected in any of our chondrocyte cultures. Most significant however was the demonstration that bGH did not dramatically affect the levels of IGFBPs in fibroblast cell cultures. We conclude that GH stimulates BP-2 production from chondrocytes and this is a cell-type specific effect in as much as it is not replicated in cultures of dermal fibroblasts.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CM conditioned medium - DMEM Dulbecco's Modified Eagles Medium - FCS foetal calf serum - IGF insulin-like growth factor - IGFBP insulin-like growth factor binding protein - HBSS Hanks' Balanced Salt Solution - GH growth hormone - NBT nitroblue tetrazolium - SFM serum free medium - TBS tris buffered saline     

Differential expression of IGF system components in proliferating vs. differentiating growth plate chondrocytes: the functional role of IGFBP-5

The growth plate is an important target tissue for insulin-like growth factors (IGFs), but little is known about the regulation of the IGF system during the developmental sequence of chondrocytes. We therefore examined the expression profile of IGF system components in proliferating vs. differentiating growth plate chondrocytes by use of two cell culture models of the growth cartilage. In rat growth plate chondrocytes in primary culture, IGF-I expression increased twofold during the process of differentiation. IGF-binding protein-3 (IGFBP-3) expression showed a biphasic pattern of with a twofold increase at the onset of differentiation and a downregulation in late differentiating chondrocytes to 25% of baseline levels; the expression patterns of IGFBP-2, -4 and -6 were not dependent on the developmental stage. In IGF- and IGFBP-3-deficient RCJ3.1C5.18 (RCJ) mesenchymal chondrogenic cells, IGFBP-2 and -6 synthesis declined by 50% during differentiation. IGFBP-5 expression was markedly upregulated during the process of differentiation in both cell culture models. Although IGFBP-5 overexpression did not have an IGF-independent effect on RCJ cell differentiation, it promoted IGF-I-enhanced differentiation of these cells. A potential mechanism for this effect is the specific increase of Akt phosphorylation in IGFBP-5-overexpressing cells in the presence of IGF-I, indicating an increased activity of the phosphatidylinositol (PI) 3-kinase pathway. These data suggest that the developmental stage of the chondrocyte is an important determinant of IGF and IGFBP expression and imply a functional role for IGFBP-5 for upregulating IGF action during chondrocyte differentiation in vivo.     

Pharmacological disruption of insulin-like growth factor 1 binding to IGF-binding proteins restores anabolic responses in human osteoarthritic chondrocytes

Insulin-like growth factor 1 (IGF-1) has poor anabolic efficacy in cartilage in osteoarthritis (OA), partly because of its sequestration by abnormally high levels of extracellular IGF-binding proteins (IGFBPs). We studied the effect of NBI-31772, a small molecule that inhibits the binding of IGF-1 to IGFBPs, on the restoration of proteoglycan synthesis by human OA chondrocytes. IGFBPs secreted by human OA cartilage or cultured chondrocytes were analyzed by western ligand blot. The ability of NBI-31772 to displace IGF-1 from IGFBPs was measured by radiobinding assay. Anabolic responses in primary cultured chondrocytes were assessed by measuring the synthesis of proteoglycans in cetylpyridinium-chloride-precipitable fractions of cell-associated and secreted ³⁵S-labeled macromolecules. The penetration of NBI-31772 into cartilage was measured by its ability to displace ¹²⁵I-labeled IGF-1 from cartilage IGFBPs. We found that IGFBP-3 was the major IGFBP secreted by OA cartilage explants and cultured chondrocytes. NBI-31772 inhibited the binding of ¹²⁵I-labeled IGF-1 to IGFBP-3 at nanomolar concentrations. It antagonized the inhibitory effect of IGFBP-3 on IGF-1-dependent proteoglycan synthesis by rabbit chondrocytes. The addition of NBI-31772 to human OA chondrocytes resulted in the restoration or potentiation of IGF-1-dependent proteoglycan synthesis, depending on the IGF-1 concentrations. However, NBI-31772 did not penetrate into cartilage explants. This study shows that a new pharmacological approach that uses a small molecule inhibiting IGF-1/IGFBP interaction could restore or potentiate proteoglycan synthesis in OA chondrocytes, thereby opening exciting possibilities for the treatment of OA and, potentially, of other joint-related diseases.     

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.     

Expression and release of insulin-like growth factor binding proteins in isolated epiphyseal growth plate chondrocytes from the ovine fetus

Insulin-like growth factor-II (IGF-II) is an autocrine modulator of epiphyseal chondrogenesis in the fetus. The cellular availability of IGFs are influenced by the IGF-binding proteins (IGFBPs). In this study, we investigated the control of expression and release of IGFBPs from isolated epiphyseal growth plate chondrocytes from the ovine fetus by hormones and growth factors implicated in the chondrogenic process. Chondrocytes were isolated from the proliferative zone of the fetal ovine proximal tibial growth plate and maintained in monolayer culture at early passage number. Culture media conditioned by chondrocytes under basal conditions released IGFBPs of 24, 34, and 29 kDa, and a less abundant species of 39-43 kDa that were identified immunologically as IGFBP-4, IGFBP-2, IGFBP-5, and IGFBP-3, respectively. Messenger RNAs encoding each species were identified by Northern blot analysis within chondrocytes, as was mRNA encoding IGFBP-6. Exposure to IGF-I or IGF-II (13 or 26 nM) caused an increase in expression and release of IGFBP-3. The release of IGFBP-2 and IGFBP-5 were also potentiated without changes to steady state mRNA, and for IGFBP-5 this was due in part to a release from the cell membrane in the presence of IGF-II. Insulin (16.7 or 167 nM) selectively increased mRNA and the release of IGFBP-3, while cortisol (1 or 5 microM) inhibited both mRNA and release of IGFBP-2 and IGFBP-5. Transforming growth factor-beta1 (TGF-beta1) (0.1 or 0.2 nM) increased the expression and release of IGFBP-3, and caused an increase in mRNAs encoding IGFBP-2 and IGFBP-5. Neither growth hormone (GH), fibroblast growth factor-2, nor thyroxine (T(4)) had any effect on IGFBP expression or release. The results suggest that IGFBP expression and release within the developing growth plate can be modulated by IGF-II and other trophic factors, thus controlling IGF availability and action.     

Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities

The insulin-like growth factor (IGF)-binding proteins (IGFBPs) are a family of six homologous proteins with high binding affinity for IGF-I and IGF-II. Information from NMR and mutagenesis studies is advancing knowledge of the key residues involved in these interactions. IGF binding may be modulated by IGFBP modifications, such as phosphorylation and proteolysis, and by cell or matrix association of the IGFBPs. All six IGFBPs have been shown to inhibit IGF action, but stimulatory effects have also been established for IGFBP-1, -3, and -5. These generally involve a decrease in IGFBP affinity and may require cell association of the IGFBP, but precise mechanisms are unknown. The same three IGFBPs have well established effects that are independent of type I IGF receptor signaling. IGFBP-1 exerts these effects by signaling through alpha(5)beta(1)-integrin, whereas IGFBP-3 and -5 may have specific cell-surface receptors with serine kinase activity. The regulation of cell sensitivity to inhibitory IGFBP signaling may play a role in the growth control of malignant cells.     

Inhibition of collagen biosynthesis and increases in low molecular weight IGF-I binding proteins in the skin of fasted rats

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen biosynthesis and prolidase activity in connective tissue cells. The disturbances in skin collagen metabolism (reflected by significant decrease in skin collagen content, collagen biosynthesis and prolidase activity) in fasted rats were accompanied by decrease in serum IGF-I level. Fasted rat serum was found to contain about 58% of IGF-I (101.6 +/- 15.4 ng/ml) as compared to control rat serum (175.7 +/- 19.8 ng/ml), while the skin of control and fasted rats contained similar concentrations of IGF-I (about 77 ng/g tissue). The insulin-like growth factor binding proteins (IGFBPs) of sera and tissue extracts (known to regulate IGF-I activity) were analysed by ligand blotting. In the serum of control rats one IGFBP band of about 46 kDa (corresponding to the acid-dissociated IGFBP-3) was detected. In the serum of fasted rats the 46 kDa IGFBP was not observed, however, an other IGFBP of about 30 kDa (corresponding to low molecular weight IGFBPs, e.g. IGFBP-1 or IGFBP-2) was found. The intensity of IGF-I binding to the 30 kDa IGFBP was much higher than that of IGFBP-3, found in control rat serum. Control and fasted rat skin contained similar IGFBPs, however their IGF-I binding abilities were much lower, compared to their serum counterparts. It was found that 46 kDa and 30 kDa proteins, observed in ligand blotting represent IGFBP-3 and IGFBP-1 or IGFBP-2. respectively as demonstrated by western immunoblot analysis. An increase in IGF-binding to 30 kDa IGFBP-1 and/or IGFBP-2 (known as an inhibitors of IGF-dependent functions) in the skin of fasted rats may explain the mechanism of reduced collagen biosynthesis and deposition in tissues during fasting.     

Stimulatory effect of a phorbol ester on expression of insulin-like growth factor (IGF) binding protein-2 and level of IGF-I receptors in mouse osteoblastic MC3T3-E1 cells

We examined the relationship between signal transduction and the expression of insulin-like growth factor I (IGF-I), IGF-I receptor level, and IGF binding proteins (IGFBPs) in murine clonal osteoblastic MC3T3-E1 cells. 12–O-Tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, decreased the secretion of immunoreactive IGF-I into the medium, whereas dibutyryl cAMP (Bt₂cAMP) augmented the secretion In contrast, TPA increased the level of type IIGF receptor on the cells. Furthermore, MC3T3-E1 cells produced and secreted at least three different IGFBPs with molecular masses of 24, 30, and 34 kDa, and the 24-kDa IGFBP was predominant under normal conditions. However, TPA specifically increased the secretion of the 34-kDa IGFBP. The N-terminal amino acid sequence of the purified 34-kDa IGFBP was nearly identical with that of rat IGFBP-2. Furthermore, the 34-kDa IGFBP was immunoreactive to anti-IGFBP-2 antiserum. The level of IGFBP-2 mRNA in the cells was increased by TPA, indicating that the increase in IGFBP-2 secretion results from the stimulation of IGFBP-2 production. In contrast, Bt₂cAMP affected neither IGF-l receptor number nor the IGFBP secretion. These results indicate that the production of IGF-l and the expression of IGF-l receptors and IGFBP-2 are up-regulated by the activation of adenylate cyclase and protein kinase C, respectively, in osteoblastic MC3T3-E1 cells. © 1994 Willey-Liss, Inc.     

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.     

Production of insulin-like growth factor binding-proteins by bovine adrenomedullary cells: differential regulation by IGF-I and dexamethasone

In the present study we examined the production of insulin-like growth factor binding proteins (IGFBPs), in chromaffin cells, a model system for sympathetic neurons. Four IGFBPs of approximately 27, approximately 31, approximately 36 and a doublet of approximately 45-50 kDa, detected in Western ligand blots of conditioned medium, were identified in Western immunoblots as IGFBP-4, IGFBP-5, IGFBP-2 and IGFBP-3, respectively. In ligand blots IGFBP-3 and IGFBP-4 appeared as the most prominent species. IGF-I (1 nM) enhanced release of IGFBP-3 while dexamethasone (1 nM) diminished release of IGFBP-4. No significant proteolytic degradation of the IGFBPs was demonstrated. Cycloheximide completely attenuated release of the IGFBPs, indicating dependency on new synthesis of the proteins. These findings are consistent with autocrine modulation of the IGF system in bovine adrenomedullary chromaffin cells by IGFBPs. Furthermore, the specific stimulatory and inhibitory effects of IGF-I and dexamethasone, respectively, on release of the predominant species of IGFBP-3 and IGFBP-4, suggested that IGFBP production may be selectively modulated in a positive and negative manner.     

Interaction of insulin-like growth factor II (IGF-II) with multiple plasma proteins: high affinity binding of plasminogen to IGF-II and IGF-binding protein-3

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of > or =150 kDa. To investigate molecular interactions between proteins involved in IGF.IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of > or =440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.     

The role of chondrocyte-matrix interactions in maintaining and repairing articular cartilage

Throughout life chondrocytes maintain the articular cartilage matrix by replacing degraded macromolecules and respond to focal cartilage injury or degeneration by increasing local synthesis activity. These observations suggest that mechanisms exist within articular cartilage that stimulate chondrocyte anabolic activity in response to matrix degradation or damage. An important cartilage anabolic factor, insulin-like growth factor I (IGF-I), appears to have a role in stimulating chondrocyte anabolic activity. Although IGF-I is ubiquitous, its bioavailability is controlled by a class of secreted proteins, IGF binding proteins (IGFPBs). Of the six known IGFPBs, IGFBP-3 is the most abundant in human articular cartilage. We recently found that with increasing age, articular chondrocytes increase their expression of IGFBP-3. This observation led us to investigate the potential role of IGFBP-3 in chondrocyte-matrix interactions. Using immunofluorescent staining and confocal microscopy we found that IGFBP-3 accumulates with increasing age in the chondrocyte territorial matrix where it co-localizes with fibronectin, but not with tenascin-C or type VI collagen. Using purified proteins we demonstrated that IGFBP-3 binds to fibronectin in a dose dependent manner, but not to tenascin-C. In vitro studies showed that IGFBP-3 alone inhibited chondrocyte synthetic activity while intact fibronectin alone significantly stimulated activity. When fibronectin and IGFBP-3 were combined we found that the inhibitory activity of low concentrations of IGFPB-3 was enhanced. These observations indicate that in mature articular cartilage IGF-I is stored in the chondrocyte territorial matrix through binding to a complex of IGFPB-3 and intact fibronectin. Storage of IGF-I of the territorial matrix may help maintain a relatively constant level of available IGF-I and the local increase in matrix synthesis following matrix damage may result from release of IGF-I. This mechanism may have an important role in maintaining and repairing articular cartilage and failure of this mechanism may lead to progressive articular cartilage degeneration.     

Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients

Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and ¹²⁵I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5–3.8 fold) and decrease in intact IGFBP-3 (0.6–0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration.     

Use of lanthanum to accurately quantify insulin-like growth factor binding to proteins on cell surfaces

Insulin-like growth factor binding proteins (IGFBPs) are found both associated with cells and in extracellular fluids. Cell-associated IGFBPs increase [¹²⁵I]-IGF binding to cell monolayers, whereas extracellular (soluble, released) IGFBPs decrease binding. In the current study, we show that either IGFBP-3 or IGFBP-5 are the major forms of IGFBP released from monolayers of human GM10 fibroblasts, T98G glioblastoma cells and forskolin-treated bovine MDBK cells. IGFBPs represent the most abundant [¹²⁵I]-IGF-I binding site on GM10 and T98G cell monolayers, but 4-17% of the total cell-associated IGFBPs are released from the cell monolayer at 8°C during their quantification. Most of the IGFBPs (> 70%) are released from MDBK cells. Quantitative estimates of [¹²⁵I]-IGF binding to the cell monolayers are altered because of the ability of the released IGFBPs to reduce the amount of radiolabeled ligand that is available to bind to the cell surface. Lanthanum (La³⁺) depresses IGFBP release from all three cell types (> 80% for GM10 and T98G cells and > 65% for MDBK cells). The effect was cation specific, noted with La³⁺ or Zn²⁺ but not with either Mn²⁺, Sr²⁺ or Se³⁺. The effect was also IGFBP specific; La³⁺ markedly depressed the release of IGFBP-3 and IGFBP-5, but had less of an effect on IGFBP-2 and IGFBP-4. Concomitant with a decrease in IGFBP-3 and IGFBP-5 release, La³⁺ caused an increase in [¹²⁵I]-IGF-I binding to cell-associated IGFBPs and type I IGF receptors. The released soluble IGFBPs have a three- to 20-fold greater affinity (K_a) for [¹²⁵I]-IGF-I compared to cell-associated IGFBPs. La³⁺ did not alter the affinity constants of cell-associated IGFBPs. In summary, we have identified a means to prevent loss of IGFBPs from cell monolayers during binding assays. This procedure will be useful in accurately quantifying the levels of IGFBPs on cell monolayers and in determining the role of cell-associated IGFBPs in controlling IGF activity. Retention of cell-associated low affinity IGFBPs may be important in controlling the size of the pericellular IGF pool and in regulating IGF-I access to the type I IGF receptor. J. Cell. Biochem. 66:256-267. © 1997 Wiley-Liss, Inc.     

Mandibular repositioning modulates IGFBP-3, -4, -5 and -6 expression in the mandibular condylar cartilage of young rats

Functional orthopedic appliances correct dental malocclusion partially by exerting indirect mechanical stimulus on the condylar cartilage, modulating growth and the adaptation of orofacial structures. However, the exact nature of the biological responses to this therapy is not well understood. Insulin-like growth factors I and II (IGF-I and II) are important local factors during growth and differentiation in the condylar cartilage [D. Hajjar, M.F. Santos and E.T. Kimura, Propulsive appliance stimulates the synthesis of insulin-like growth factors I and II in the mandibular condylar cartilage of young rats, Arch. Oral Biol. 48 (2003), 635-642]. The bioefficacy of IGFs at the cellular level is modulated by IGF binding proteins (IGFBP). The aim of this study was to verify the mRNA and protein expression of IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 in the condylar cartilage of young male Wistar rats that used a mandibular propulsive appliance for 3, 9, 15, 20, 30 or 35 days. For this purpose, sagittal sections of decalcified and paraffin-embedded condyles were submitted to immunohistochemistry and the condylar cartilage to RT-PCR. The control group showed a gradual increase in the protein expression of all IGFBPs, except IGFBP-4. Following use of the appliance, IGFBP-3 and IGFBP-6 expression decreased in the early stage of the treatment. At 20 days of treatment there was a decline in the IGFs and IGFBP-3, IGFBP-4 and IGFBP-5 expression and at 30 days there was a peak in the IGFs and all IGFBPs expression except for IGFBP-3 where the peak was observed in the control animals. The expression patterns of all IGFBPs in the condylar cartilage were similar. The modulation of IGFBP-3, -4, -5 and -6 expression in the condylar cartilage in response to the propulsive appliance suggests that those peptides are involved in the mandibular adaptation during this therapy.     

Endogenous IGF-I regulates IGF binding protein production in human intestinal smooth muscle cells

Human intestinal smooth muscle in culture produces insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3, IGFBP-4, and IGFBP-5, which modulate the effects of IGF-I. This study examined the regulation of IGFBP production by endogenous IGF-I. R3-IGF-I, an agonist unaffected by IGFBPs, elicited concentration-dependent increase in growth, measured by [(3)H]thymidine incorporation, and production of IGFBP-3, IGFBP-4, and IGFBP-5, measured by Western blot. Antagonists of the IGF-I receptor, IGF-I Analog or monoclonal antibody 1H7, elicited concentration-dependent inhibition of growth and decrease in IGFBP-3, IGFBP-4, and IGFBP-5 production, implying that endogenous IGF-I stimulated growth and IGFBP production. R3-IGF-I-induced increase in IGFBP-3, IGFBP-4, and IGFBP-5 production was partially inhibited by a mitogen-activated protein (MAP) kinase or a phosphatidylinositol-3-kinase (PI 3-kinase) inhibitor and abolished by the combination. We conclude that endogenous IGF-I stimulates growth and IGFBP-3, IGFBP-4, and IGFBP-5 production in human intestinal smooth muscle cells. Regulation of IGFBP production by IGF-I is mediated by activation of distinct MAP kinase and PI 3-kinase pathways, the same pathways through which IGF-I stimulates growth.     

Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rlGFBP-1, 2, or 3.

The bioavailability and action of the insulin-like growth factors (IGFs) are determined by specific IGF-binding proteins (IGFBP) to which they are complexed. Complementary DNA clones have been isolated that encode three related IGFBPs: human IGFBP-1 (hIGFBP-1), human IGFBP-3 (hIGFBP-3), and rat IGFBP-2 (rIGFBP-2). IGFBP-1 and IGFBP-3 are regulated differently in human plasma, suggesting that they have different functions. In order to study the molecular basis of the regulation of the different IGFBPs, we have identified a panel of rat cell lines that express a single predominant binding protein and developed an assay strategy to distinguish the different binding proteins. Proteins in conditioned medium were examined by ligand blotting, and by immunoprecipitation and immunoblotting using antibodies to rIGFBP-2 and hIGFBP-1; RNAs were hybridized to cDNA probes for rIGFBP-2 and hIGFBP-1. 1) C6 glial cells and B104 neuroblastoma cells express an approximately 40 kilodalton (kDa) glycosylated binding protein that most likely represents rIGFBP-3, the binding subunit of the 150 kDa IGF: binding protein complex in adult rat serum. The C6 and B104 binding proteins do not react with antibodies to rIGFBP-2, and RNAs from C6 and B104 cells do not hybridize to cDNA probes for rIGFBP-2 or hIGFBP-1. 2) BRL-3A, Clone 9, and TRL 12-15 cell lines derived from normal rat liver express rIGFBP-2, a 30 kDa nonglycosylated IGF-binding protein that is recognized by antibodies to rIGFBP-2 but not by antibodies to hIGFBP-1. RNAs from these cells hybridize to a rIGFBP-2 cDNA probe, but not to a hIGFBP-1 probe. 3) H35 rat hepatoma cells express a 30 kDa nonglycosylated IGFBP that is presumptively identified as rIGFBP-1. It does not react with antibodies to rIGFBP-2, but is recognized by polyclonal and monoclonal antibodies to hIGFBP-1. RNA from H35 cells hybridizes to a hIGFBP-1 cDNA probe, but not to a rIGFBP-2 probe. Expression of rIGFBP-1 by the H35 cell line has enabled us to establish and validate specific assays for this protein that allow us to study its regulation in intact rats. Identification of a panel of rat cell lines expressing specific IGFBPs should be useful in elucidating the molecular mechanisms of IGFBP regulation.