Vitamin D3 metabolites regulate LTBP1 and latent TGF-beta1 expression and latent TGF-beta1 incorporation in the extracellular matrix of chondrocytes

Growth plate chondrocytes make TGF-beta1 in latent form (LTGF-beta1) and store it in the extracellular matrix via LTGF-beta1 binding protein (LTBP1). 1,25-(OH)2D3 (1,25) regulates matrix protein production in growth zone (GC) chondrocyte cultures, whereas 24,25-(OH)2D3 (24,25) does so in resting zone (RC) cell cultures. The aim of this study was to determine if 24,25 and 1,25 regulate LTBP1 expression as well as the LTBP1 -mediated storage of TGF-beta1 in the extracellular matrix of RC and GC cells. Expression of LTBP1 and TGF-beta1 in the growth plate and in cultured RC and GC cells was determined by in situ hybridization using sense and antisense oligonucleotide probes based on the published rat LTBP1 and TGF-beta1 cDNA sequences. Fourth passage male rat costochondral RC and GC chondrocytes were treated for 24 h with 10(-7)-10(-9) M 24,25 and 10(-8)-10(-10) M 1,25, respectively. LTBP1 and TGF-beta1 mRNA levels were measured by in situ hybridization; production of LTGF-beta1, LTGF-beta2, and LTBP1 protein in the conditioned media was verified by immunoassays of FPLC-purified fractions. In addition, ELISA assays were used to measure the effect of 1,25 and 24,25 on the level of TGF-beta1 in the media and matrix of the cultures. Matrix-bound LTGF-beta1 was released by digesting isolated matrices with 1 U/ml plasmin for 3 h at 37 degrees C. LTBP1 and TGF-beta1 mRNAs are co-expressed throughout the growth plate, except in the lower hypertrophic area. Cultured GC cells express more LTBP1 and TGF-beta1 mRNAs than RC cells. FPLC purification of the conditioned media confirmed that RC cells produce LTGF-beta1, LTGF-beta2, and LTBP1. GC cells also produce LTGF-beta2, but at lower concentrations. 1,25 dose-dependently increased the number of GC cells with high LTBP1 expression, as seen by in situ hybridization. 24,25 had a similar, but less pronounced, effect on RC cells. 1,25 also caused a dose-dependent increase in the amount of TGF-beta1 protein found in the matrix, significant at 10(-8) and 10(-9) M, and a corresponding decrease in TGF-beta1 in the media. 24,25 had no effect on the level of TGF-beta1 in the matrix or media produced by RC cells. This indicates that 1,25 induces the production of LTBP1 by GC cells and suggests that the TGF-beta1 content of the media is reduced through the formation of latent TGF-beta1 -LTBP1 complexes which mediates storage in the matrix. Although 24,25 induced the expression of LTBP1 by RCs, TGF-beta1 incorporation into the matrix is not regulated by this vitamin D3 metabolite. Thus, vitamin D3 metabolites may play a role in regulating the availability of TGF-beta1 by modulating LTBP1 production.     

Insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor mediates paracrine interactions during spermatogonial development

The insulin-like growth factor-II/cation-independent mannose 6-phosphate (IGF-II/M6P) receptor transduces signals after binding IGF-II or M6P-bearing growth factors. We hypothesized that this receptor relays paracrine signals between Sertoli cells and spermatogonia in the basal compartment of the seminiferous epithelium. For these studies spermatogonia were isolated from 8-day-old mice with purity >95% and viability >85% after overnight culture. The IGF-II/M6P receptors were present on the surface of spermatogonia, as detected by indirect immunofluorescence. We determined that both IGF-II and M6P-glycoproteins in Sertoli cell conditioned medium (SCM) modulate gene expression in isolated spermatogonia. The IGF-II produced dose-dependent increases in both rRNA and c-fos mRNA. These effects were mediated specifically by IGF-II/M6P receptors, as shown by studies using IGF-II analogues that are specific agonists for either IGF-I or IGF-II receptors. The SCM treatment also induced dose-dependent increases in rRNA levels, and M6P competition showed that this response required interaction with IGF-II/M6P receptors. The M6P-glycoproteins isolated from SCM by IGF-II/M6P receptor affinity chromatography increased spermatogonial rRNA levels at much lower concentrations than required by SCM treatment, providing further evidence for the paracrine activity of Sertoli M6P-glycoproteins. These results demonstrate that Sertoli cells secrete paracrine factors that modulate spermatogonial gene expression after interacting with cell-surface IGF-II/M6P receptors.     

Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self- regulating system

The conversion of latent transforming growth factor beta (LTGF-beta) to the active species, transforming growth factor beta (TGF-beta), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF-beta in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-beta as determined by this assay. The concentration of TGF-beta in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A+ RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-beta 1 and TGF-beta 2, while SMCs produced primarily TGF-beta 1. No change in the expression of these two forms of TGF-beta was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-beta indicated that most of the active TGF-beta was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-beta in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-beta in the co-culture medium blocked the observed increase in PAI-1 levels. The increased expression of PAI-1 subsequent to TGF-beta formation blocked the activation of the protease required for conversion of LTGF-beta to TGF-beta as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-beta. This effect was lost upon removal of the PAI-1 antibodies. Thus, the activation of LTGF-beta appears to be a self-regulating system.     

Interactions of recombinant and platelet transforming growth factor-beta 1 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor

Recombinant transforming growth factor (TGF)-beta 1 precursor was recently found to contain mannose 6-phosphate (Purchio et al., 1988, J. Biol. Chem. 263, 14211-14215). In the present study, recombinant TGF-beta 1 precursor was shown to bind to the insulin-like growth factor (IGF)-II/mannose 6-phosphate (man6P) receptor on the plasma membrane of cells since: 1) Insulin, which induces an increase in cell surface IGF-II/man6P receptors on adipocytes, caused a 2.7-fold increase in TGF-beta 1 precursor binding to adipocytes; 2) Chinese hamster ovary cells selected for overexpression of the IGF-II/man6P receptor exhibited an increased binding of TGF-beta 1 precursor in comparison to the parental cells; and 3) the binding of 125I-TGF-beta 1 precursor to these transfected cells and adipocytes was largely inhibited by man6P. After 15 minutes at 37 degrees C, 75% of the recombinant TGF-beta 1 precursor was found to be internalized in the transfected cells. Additional studies with latent TGF-beta 1 isolated from platelets indicated that this material could also bind to the isolated IGF-II/man6P receptor.     

Loss of insulin-like growth factor II receptor expression promotes growth in cancer by increasing intracellular signaling from both IGF-I and insulin receptors

The insulin-like growth factor-II receptor (IGF-IIR) is frequently mutated or deleted in some malignant human tumors, suggesting that the IGF-IIR is a tumor suppressor. However, the exact mechanism by which IGF-IIR suppresses growth in tumors has not been definitively established. We demonstrate that IGF-IIR-deficient murine L cells (D9) have higher growth rates than IGF-IIR-positive L cells (Cc2) in response to IGF-II. IGF-II levels are higher in growth-conditioned medium from D9 versus Cc2 cells. Receptor neutralization studies and measurements of insulin receptor substrate 1 phosphorylation confirm that the enhanced growth of D9 cells is due to increased stimulation of the IGF-I and insulin receptors by IGF-II. In contrast, the levels of secreted latent and active transforming growth factor beta (TGF-beta) are similar for both D9 and Cc2 cells, indicating that the slower growth of Cc2 cells is not due to activation of latent TGF-beta by IGF-IIR and growth inhibition. The results directly demonstrate that down regulation of the IGF-IIR promotes the growth of transformed D9 cells by sustaining IGF-II, which binds to and activates IGF-IR and insulin receptor to increase intracellular growth signals.     

The mechanism for the activation of latent TGF-beta during co-culture of endothelial cells and smooth muscle cells: cell-type specific targeting of latent TGF-beta to smooth muscle cells

Transforming growth factor-beta (TGF-beta) is secreted in a latent form and activated during co-culture of endothelial cells and smooth muscle cells. Plasmin located on the surface of endothelial cells is required for the activation of latent TGF-beta (LTGF-beta) during co-culture, and the targeting of LTGF-beta to the cellular surface is requisite for its activation. In the present study, the cellular targeting of LTGF- beta was examined. We detected the specific binding of 125I-large LTGF- beta 1 isolated from human platelets to smooth muscle cells but not to endothelial cells. A mAb against the latency-associated peptide (LAP) of large LTGF-beta 1 complex, which blocked the binding of 125I-large LTGF-beta 1 to smooth muscle cells, inhibited the activation of LTGF- beta during co-culture. The binding of 125I-large LTGF-beta 1 could not be competed either by mannose-6-phosphate (300 microM) or by the synthetic peptide Arg-Gly-Asp-Ser (300 micrograms/ml). These results indicate that the targeting of LTGF-beta to smooth muscle cells is required for the activation of LTGF-beta during co-culture of endothelial cells and smooth muscle cells. The targeting of LTGF-beta to smooth muscle cells is mediated by LAP, and the domain of LAP responsible for the targeting to smooth muscle cells may not be related to mannose-6-phosphate or an Arg-Gly-Asp sequence, both of which have been previously proposed as candidates for the cellular binding domains within LAP.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

Isoform-specific activation of latent transforming growth factor beta (LTGF-beta) by reactive oxygen species

The three mammalian transforming growth factor beta (TGF-beta) isoforms are each secreted in a latent complex in which TGF-beta homodimers are non-covalently associated with homodimers of their respective pro-peptide called the latency-associated peptide (LAP). Release of TGF-beta from its LAP, called activation, is required for binding of TGF-beta to cellular receptors, making extracellular activation a critical regulatory point for TGF-beta bioavailability. Our previous work demonstrated that latent TGF-beta1 (LTGF-beta1) is efficiently activated by ionizing radiation in vivo and by reactive oxygen species (ROS) generated by Fenton chemistry in vitro. In the current study, we determined the specific ROS and protein target that render LTGF-beta1 redox sensitive. First, we compared LTGF-beta1, LTGF-beta2 and LTGF-beta3 to determine the generality of this mechanism of activation and found that redox-mediated activation is restricted to the LTGF-beta1 isoform. Next, we used scavengers to determine that ROS activation was a function of OH(.) availability, confirming oxidation as the primary mechanism. To identify which partner of the LTGF-beta1 complex was functionally modified, each was exposed to ROS and tested for the ability to form a latent complex. Exposure of TGF-beta1 did not alter its ability to associate with LAP, but exposing LAP-beta1 to ROS prohibited this phenomenon, while treatment of ROS-exposed LAP-beta1 with a mild reducing agent restored its ability to neutralize TGF-beta1 activity. Taken together, these results suggest that ROS-induced oxidation in LAP-beta1 triggers a conformational change that releases TGF-beta1. Using site-specific mutation, we identified a methionine residue at amino acid position 253 unique to LAP-beta1 as critical to ROS-mediated activation. We propose that LTGF-beta1 contains a redox switch centered at methionine 253, which allows LTGF-beta1 to act uniquely as an extracellular sensor of oxidative stress in tissues.     

The mannose 6-phosphate/insulin-like growth factor-II receptor is a substrate of type V transforming growth factor-beta receptor.

The type V transforming growth factor beta (TGF-beta) receptor (TbetaR-V) is a ligand-stimulated acidotropic Ser-specific protein kinase that recognizes a motif of SXE/S(P)/D. This motif is present in the cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor. We have explored the possibility that the Man-6-P/IGF-II receptor is a substrate of TbetaR-V. Purified bovine Man-6-P/IGF-II receptor was phosphorylated by purified bovine TbetaR-V in the presence of [gamma-32P]ATP and MnCl2 with an apparent Km of 130 nM. TGF-beta stimulated the phosphorylation of the Man-6-P/IGF-II receptor at 0 degrees C in mouse L cells overexpressing the Man-6-P/IGF-II receptor and in wild-type mink lung epithelial (Mv1Lu cells) metabolically labeled with [32P]orthophosphate. The in vitro and in vivo phosphorylation of the Man-6-P/IGF-II receptor occurred at the putative phosphorylation sites as revealed by phosphopeptide mapping and amino acid sequence analysis. TGF-beta stimulated Man-6-P/IGF-II receptor-mediated uptake (approximately 2-fold after 12 h treatment) of exogenous beta-glucuronidase in Mv1Lu cells and type II TGF-beta receptor (TbetaR-II)-defective mutant cells (DR26 cells) but not in type I TGF-beta receptor (TbetaR-I)-defective mutant cells (R-1B cells) and human colorectal carcinoma cells (RII-37 cells) expressing TbetaR-I and TbetaR-II but lacking TbetaR-V. These results suggest the Man-6-P/IGF-II receptor serves as an in vitro and in vivo substrate of TbetaR-V and that both TbetaR-V and TbetaR-I may play a role in mediating the TGF-beta-stimulated uptake of exogenous beta-glucuronidase.     

Interactions of cathepsin-D and insulin-like growth factor-II (IGF-II) on the IGF-II/mannose-6-phosphate receptor in human breast cancer cells and possible consequences on mitogenic activity of IGF-II

The lysosomal enzyme cathepsin-D (cath-D) and insulin-like growth factor-II (IGF-II), which share a common IGF-II/mannose-6-phosphate (M6P) transmembrane receptor, are both synthesized and secreted by breast cancer cells, upon which they might exert an intracrine/autocrine control on proliferation. We have evaluated the binding of 125I-immunopurified human cath-D in different breast cell membrane preparations. The concentration of high affinity M6P reversible binding sites (mean Kd, 0.85 nM) varied among the different breast cancer cells (0-0.82 pmol/mg membrane protein), but there was no correlation between the presence of steroid receptor and M6P-dependent binding. Cross-linking experiments with [125I]cath-D and [125I]IGF-II showed the formation of complexes with the 270,000 mol wt IGF-II/M6P receptor molecule which migrated, respectively, at 330,000 and 270,000 mol wt in 3-10% gradient sodium dodecyl sulfate-polyacrylamide gels. [125I]IGF-II cross-linking was increased by M6P (20% above control), whereas cath-D strongly inhibited IGF-II interaction by 80%. Conversely, IGF-II reduced [125I]cath-D cross-linking by 55%. Direct ligand binding on receptors transferred onto nitrocellulose sheets by Western blotting confirmed the interaction of both ligands on the same receptor molecule. By studying IGF-II's growth-promoting activity in these cells in a wide range of concentrations, we show that IGF-II triggers its mitogenic response via IGF-II/M6P receptor at low concentrations, whereas it is mainly acting via IGF-I receptor at high concentrations. Three lines of evidences lead us to that conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoprotein (a) inhibits the generation of transforming growth factor beta: an endogenous inhibitor of smooth muscle cell migration

Conditioned medium (CM) derived from co-cultures of bovine aortic endothelial cells (BAECs) and bovine smooth muscle cells (BSMCs) contains transforming growth factor-beta (TGF-beta) formed via a plasmin-dependent activation of latent TGF-beta (LTGF beta), which occurs in heterotypic but not in homotypic cultures (Sato, Y., and D. B. Rifkin. 1989. J. Cell Biol. 107: 1199-1205). The TGF-beta formed is able to block the migration of BSMCs or BAECs. We have found that the simultaneous addition to heterotypic culture medium of plasminogen and the atherogenic lipoprotein, lipoprotein (a) (Lp(a)), which contains plasminogen-like kringles, inhibits the activation of LTGF-beta in a dose-dependent manner. The inclusion of LDL in the culture medium did not show such an effect. Control experiments indicated that Lp(a) does not interfere with the basal level of cell migration, the activity of exogenous added TGF-beta, the release of LTGF-beta from cells, the activation of LTGF-beta either by plasmin or by transient acidification, or the activity of plasminogen activator. The addition of Lp(a) to the culture medium decreased the amount of plasmin found in BAECs/BSMCs cultures. Similar results were obtained using CM derived from cocultures of human umbilical vein endothelial cells and human foreskin fibroblasts. These results suggest that Lp(a) can inhibit the activation of LTGF-beta by competing with the binding of plasminogen to cell or matrix surfaces. Therefore, high plasma levels of Lp(a) might enhance smooth muscle cell migration by decreasing the levels of the migration inhibitor TGF-beta thus contributing to generation of the atheromatous lesions.     

Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells

A hitherto unknown function for transglutaminase (TGase; R-glutaminyl- peptide: amine gamma-glutamyltransferase, EC 2.3.2.13) was found in the conversion of latent transforming growth factor-beta (LTGF-beta) to active TGF-beta by bovine aortic endothelial cells (BAECs). The cell- associated, plasmin-mediated activation of LTGF-beta to TGF-beta induced either by treatment of BAECs with retinoids or by cocultures of BAECs and bovine smooth muscle cells (BSMCs) was blocked by seven different inhibitors of TGase as well as a neutralizing antibody to bovine endothelial cell type II TGase. Control experiments indicated that TGase inhibitors and/or a neutralizing antibody to TGase did not interfere with the direct action of TGF-beta, the release of LTGF-beta from cells, or the activation of LTGF-beta by plasmin or by transient acidification. After treatment with retinoids, BAECs expressed increased levels of TGase coordinate with the generation of TGF-beta, whereas BSMCs and bovine embryonic skin fibroblasts, which did not activate LTGF-beta after treatment with retinoids, did not. Furthermore, both TGase inhibitors and a neutralizing antibody to TGase potentiated the effect of retinol in enhancing plasminogen activator (PA) levels in cultures of BAECs by suppressing the TGF-beta-mediated enhancement of PA inhibitor-1 (PAI-1) expression. These results indicate that type II TGase is a component required for cell surface, plasmin-mediated LTGF-beta activation process and that increased expression of TGase accompanies retinoid-induced activation of LTGF- beta.     

Insulin-like growth factor-II/mannose 6 phosphate receptors facilitate the matrix effects of latent transforming growth factor-β1 released from genetically modified keratinocytes in a fibroblast/keratinocyte co-culture system

This study was conducted to explore the mechanism of activation of transforming growth factor-β1 (TGF-β1) which is critical to its role in many physiological and pathological conditions. To date, almost all reports concerning TGF-β1 activation delineated that release of mature TGF-β1 from latency associated protein (LAP) is required for its activation. We report that latent TGF-β1 (LTGF-β1) released from TGF-β1 genetically modified keratinocytes grown in the top chamber of a co-culture system functions as a fibrogenic factor through interaction with insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors of human dermal fibroblasts grown in the lower chamber of this system. Following successful transduction, the pLin-LTGF-β1 vector was amplified in PA317 packaging cells which possess viral structural proteins for vector in the presence of neomycin. Conditioned medium derived from packaging cells containing competent viral particles was then used to transduce either keratinocytes or fibroblasts grown in the upper chamber of a co-culture system, in which a 0.4 μm porous membrane separates the two chambers. In this way, LTGF-β1 produced by transduced cells in the upper chamber is released and diffuses into the lower chambers where dermal fibroblasts are grown. Conditioned medium from the lower chamber was removed 3 days later and used to evaluate the latency and bioactivity of TGF-β1 using enzyme-linked immunosorbent assay (ELISA) and mink lung (Mv1Lu) epithelial growth inhibition assay. Cells were also harvested and used for RNA extraction. The results of these experiments showed that 1) the TGF-β1-LAP complex, which was latent in traditionally used mink lung growth inhibition assay, directly modulated the expression of collagenase, type I, and type III collagen mRNA by dermal fibroblasts; 2) this stimulation was inhibited by M6P in a dose-dependent manner; 3) the TGF-β1-LAP inhibits Mv1Lu epithelial cells only when this complex was incubated with cell membranes isolated from dermal fibroblasts; and 4) LTGF-β1 activation seems to occur through a conformational alteration rather than by release of the mature TGF-β1 from LAP in our co-cultured system. This conformational alteration seems to occur through the interaction of the TGF-β1-LAP complex with the IGF-II/M6P receptors. Thus, the quantity of IGF-II/M6P receptors is important in cellular response to LTGF-β1 in any physiological and pathological conditions. J. Cell. Physiol. 180:61–70, 1999. © 1999 Wiley-Liss, Inc.     

Phospholipase C and protein kinase C-β 2 mediate insulin-like growth factor II-dependent sphingosine kinase 1 activation

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.     

Modulation of insulin-like growth factor-II/mannose 6-phosphate receptors and transforming growth factor-beta 1 during liver regeneration.

Transforming growth factor-beta 1 (TGF-beta 1) is a potent mito-inhibiting substance that is thought to play an important function in regulating hepatocyte proliferation during liver regeneration. In this investigation, we have shown by immunohistochemistry that hepatocytes containing significant intracellular concentrations of TGF-beta 1 12 h after a two-thirds partial hepatectomy. This increase in hepatocyte TGF-beta 1 concentration was initially confined to those cells that resided in the periportal region of the liver. The elevation of intracellular TGF-beta 1 was, however, transient, and within 36 h, the hepatocytes positive for TGF-beta 1 had changed in a wavelike fashion from the periportal to the pericentral region of the liver lobules. By 48 h, most hepatocytes no longer contained TGF-beta 1. Interestingly, this temporary increase in TGF-beta 1 always preceded the onset of hepatocyte replication by approximately 3-6 h. Since TGF-beta 1 mRNA has been shown to be absent from hepatocytes normally and throughout liver regeneration, these results imply that the increase in intracellular TGF-beta 1 resulted from an augmented uptake. We have further shown that the insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man-6-P) receptors were up-regulated during liver regeneration and that the increased expression of this receptor co-localized in those hepatocytes containing elevated concentrations of TGF-beta 1. The latent TGF-beta 1 phosphomannosyl glycoprotein complex has been shown to bind to the IGF-II/Man-6-P receptor. Therefore, our data are consistent with the hypothesis that this latent complex is internalized through the IGF-II/Man-6-P receptor to the intracellular acidic prelysosomal/endosomal compartments where the mature TGF-beta 1 molecule could be activated by dissociation from the latent complex.     

Trans-10,cis-12-conjugated linoleic acid inhibits Caco-2 colon cancer cell growth

A commercially available mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer cell growth. We compared the individual potencies of the two main isomers in this mixture [cis-9,trans-11 (c9t11) and trans-10,cis-12 (t10c12)] and assessed whether decreased cell growth is related to changes in secretion of insulin-like growth factor II (IGF-II) and/or IGF-binding proteins (IGFBPs), which regulate Caco-2 cell proliferation. Cells were incubated in serum-free medium with different concentrations of the individual CLA isomers. t10c12 CLA dose dependently decreased viable cell number (55 +/- 3% reduction 96 h after adding 5 microM t10c12 CLA). t10c12 CLA induced apoptosis and decreased DNA synthesis, whereas c9t11 CLA had no effect. Immunoblot analysis of 24-h serum-free conditioned medium using a monoclonal anti-IGF-II antibody revealed that Caco-2 cells secreted both a mature 7,500 molecular weight (M(r)) IGF-II and higher M(r) forms of IGF-II. The levels of the higher M(r) and the mature form of IGF-II were decreased 50 +/- 3% and 22 +/- 2%, respectively, by 5 microM t10c12 CLA. c9t11 CLA had no effect. Ligand blot analysis of conditioned medium using 125I-labeled IGF-II revealed that t10c12 CLA slightly decreased IGFBP-2 production; c9t11 CLA had no effect. Exogenous IGF-II reversed t10c12 CLA-induced growth inhibition and apoptosis. These results indicate that CLA-inhibited Caco-2 cell growth is caused by t10c12 CLA and may be mediated by decreasing IGF-II secretion in Caco-2 cells.     

Expression of IGF receptors on alveolar macrophages: IGF-induced changes in InsPi formation, [Ca2+]i, and pHi

Expression of insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose-6-phosphate (IGF-II/Man6P) receptors in cultured bovine alveolar macrophages (BAM) was demonstrated by competitive binding studies and crosslinking experiments. Western blotting of protein extracts from cultured BAM using an anti bovine IGF-II/Man6P receptor antiserum (#66416) confirmed the presence of IGF-II/Man6P receptors on BAM. The effects of IGFs and Man6P on generation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[³H]inositol-labelled cultured BAM. IGF-I at nanomolar concentrations and Man6P (10^-8–10³ M) stimulated the accumulation of both Ins(1,4,5)P₃ and Ins(1,3,4,5)P₄ after 30 sec. IGF-II (up to 2.3 × 10^-8M) had no significant effect on inositol phosphate accumulation under the same conditions. Both IGFs and Man6P induced a rise in [Ca²⁺]_iin cultured BAM. In addition, using the fluorescent dye SNARF-1/AM we could demonstrate rapid but small IGF-II (10^-9 M) triggered acidification (0.07 pH units) of cultured BAM. Taken together, our results indicate not only the presence of both IGF-I and IGF-II/Man6P receptors on BAM, but also provide evidence of the linkage of the IGF-I receptor to the inositol phosphate system.     

Heterotrimeric G Proteins and the Single-Transmembrane Domain IGF-II/M6P Receptor: Functional Interaction and Relevance to Cell Signaling

The G protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Classical GPCR signaling constitutes ligand binding to a seven-transmembrane domain receptor, receptor interaction with a heterotrimeric G protein, and the subsequent activation or inhibition of downstream intracellular effectors to mediate a cellular response. However, recent reports on direct, receptor-independent G protein activation, G protein-independent signaling by GPCRs, and signaling of nonheptahelical receptors via trimeric G proteins have highlighted the intrinsic complexities of G protein signaling mechanisms. The insulin-like growth factor-II/mannose-6 phosphate (IGF-II/M6P) receptor is a single-transmembrane glycoprotein whose principal function is the intracellular transport of lysosomal enzymes. In addition, the receptor also mediates some biological effects in response to IGF-II binding in both neuronal and nonneuronal systems. Multidisciplinary efforts to elucidate the intracellular signaling pathways that underlie these effects have generated data to suggest that the IGF-II/M6P receptor might mediate transmembrane signaling via a G protein-coupled mechanism. The purpose of this review is to outline the characteristics of traditional and nontraditional GPCRs, to relate the IGF-II/M6P receptor’s structure with its role in G protein-coupled signaling and to summarize evidence gathered over the years regarding the putative signaling of the IGF-II/M6P receptor mediated by a G protein.