Transforming growth factor-beta induces growth inhibition and IGF-binding protein-3 production in prostatic stromal cells: abnormalities in cells cultured from benign prostatic hyperplasia tissues.

The IGF axis has been implicated in the pathogenesis of benign prostatic hyperplasia (BPH) via the paracrine action of IGFs and IGF-binding proteins (IGFBPs). In this study, we examined the regulation of cell growth and IGFBP-3 secretion by transforming growth factor-beta (TGF-beta) in prostatic stromal cell (PC-S) cultures from histologically normal tissues and tissues from BPH. PC-S cultures were treated with varying doses of TGF-beta1. Forty-eight hour conditioned media (CM) from these cultures were subjected to Western immunoblotting and ligand blotting for detection and quantification of IGFBPs. IGFBPs-2, -3 and -4 were detected in the CM from normal PC-S cultures. In CM from BPH PC-S, IGFBP-3 levels were 2-fold lower at baseline than in the normal PC-S CM, in addition to the differences in IGFBPs-2 and -5 which we have previously reported. In response to TGF-beta1, a 15-fold increase in the levels of IGFBP-3 was observed in normal PC-S CM, while a mere 2-fold increase was observed in BPH PC-S CM (P<0.001). These findings were confirmed by specific immunoblotting and immunocytochemistry. IGFBP-3 mRNA levels detected by Northern blotting of total RNA extracted from similar cultures showed the induction of IGFBP-3 expression by TGF-beta1 in normal PC-S and its lack of induction in BPH PC-S. Cell growth inhibition in response to TGF-beta1 correlated with the IGFBP-3 concentrations found in CM. Normal PC-S showed a 60% decrease in cell number after 10 days in media with 1 ng/ml TGF-beta1, compared with the untreated control. The decrease in proliferation observed in comparably treated BPH cells was only 20% (P<0.001). In conclusion, BPH PC-S had a reduced IGFBP-3 response to TGF-beta1 and demonstrated decreased TGF-beta1-induced growth inhibition relative to normal PC-S. We hypothesize that in normal PC-S, TGF-beta exerts its anti-proliferative effects by stimulating the production of IGFBP-3, which acts as an inhibitory factor, either by inhibiting IGFs or directly by interacting with cells, and that this process is altered in BPH PC-S.

Characterization and chromosomal mapping of a mouse ortholog of the late-infantile ceroid-lipofuscinosis gene CLN2.

Late-infantile ceroid-lipofuscinosis (CLN2) is an autosomal recessively inherited, neurodegenerative disease in humans. The CLN2 locus has been mapped to Chromosome (Chr) 11p15, and its sequence and genomic organization have recently been reported. In the present study, the cDNA sequence, exon/intron organization, and chromosomal localization of a mouse ortholog of the CLN2 gene are described. The mouse cDNA contains an open reading frame that predicts a protein product of 562 amino acids. The mouse and human coding regions are 86% and 88% identical at the nucleic acid and amino acid levels, respectively. One less codon appears in the mouse cDNA when compared with the human ortholog. The mouse gene (Cln2) spans more than 6 kb and consists of 13 exons separated by introns ranging in size from 111 to 1259 bp. Length polymorphism in an (AC)(n) microsatellite in intron 3 of the mouse Cln2 gene was used to perform segregation analysis with The Jackson Laboratory DNA Panel Mapping Resource. On the basis of this analysis, the Cln2 gene was localized to a region of mouse Chr 7 that corresponds to human Chr 11p15. Characterization of the mouse Cln2 gene will facilitate generation of a mouse model for late-infantile ceroid-lipofuscinosis by gene targeting and identification of functionally important regions of the Cln2 protein.

Identification of novel high molecular weight insulin-like growth factor-binding protein-3 association proteins in human serum.

The insulin-like growth factor (IGF)-binding proteins (IGFBPs) carry IGFs in serum and regulate their activity and bioavailability. The main IGFBP in serum, IGFBP-3, is known to form a 150-kDa complex with IGFs and the acid-labile subunit (ALS). We investigated the binding of IGFBP-3 to additional association proteins in human serum (IGFBP-3 APs). Ligand blots, column chromatography, and affinity cross-linking experiments revealed the specific binding of IGFBP-3 to at least three novel serum proteins. These techniques demonstrated the presence of proteins with molecular masses of 70, 100, and 150 kDa that bind IGFBP-3 with high affinity. Serum ALS migrated separately (at 88 kDa) from the novel IGFBP-3 APs (as evident by Western immunoblot), and bound IGFBP-3 weakly (by reverse ligand blots). We also demonstrated that large amounts of one of the IGFBP-3 APs and small amounts of ALS were coimmunoprecipitated with IGFBP-3 from human serum. Similar to ALS, these IGFBP-3 APs are acid labile and lose their IGFBP-3 binding capacity after exposure to low pH. We conclude that there are several serum proteins in addition to ALS and IGFs that bind IGFBP-3 with high affinity. These IGFBP-3 APs may serve as an additional reservoir of IGFBP-3 or modulate its functions.

Acid-activated insulin-like growth factor binding protein protease activity of cathepsin D in normal and malignant prostatic epithelial cells and seminal plasma.

In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1-6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiological characteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferation and invasive tumor growth.

IGF-II, IGF-binding proteins and IGF receptors in pancreatic beta-cell lines.

The IGFs are mitogenic agents which are closely linked to regulatory processes in carbohydrate metabolism. Because limited information is available on the occurrence of the IGF system in the pancreatic beta-cell milieu, we evaluated the presence of IGFs, IGF receptors, and IGF-binding proteins (IGFBPs) in the beta-cell lines beta TC3 and HIT T-15. Serum-free conditioned media (SFCM) from beta TC3 cells contained IGF-II at concentrations greater than 100 ng/ml. High (15 kDa) and low (7.5 kDa) molecular weight IGF-II were detected both by column chromatography followed by RIA and by immunoblotting. GH (10-1000 ng/ml) conditioning of beta TC3 cells stimulated IGF-II secretion in a dose-dependent manner. IGF-II mRNA was detected in beta TC3 cells using Northern blots, and also showed a GH-dependent relationship. IGF-II peptide was detected in SFCM from HIT cells, albeit at lower concentrations. To evaluate the presence of IGF receptors in beta-cell lines, affinity cross-linking studies were performed on beta TC3 cells, demonstrating type I IGF receptors which bound iodinated IGF-II with high affinity, iodinated IGF-I with lesser affinity, and had minimal appreciable binding to iodinated insulin. Type II IGF receptors were not detected. SFCM from beta TC3 and HIT cells was subjected to Western ligand blotting, which disclosed the presence of two major IGFBPs of 29 kDa and 24 kDa, characteristic of IGFBP-2 and IGFBP-4. The identity of the specific IGFBPs was confirmed by immunoprecipitation and Northern blotting. Varying the glucose concentration had no significant effect on the levels of IGFBPs, nor did preconditioning with GH, IGF-I, IGF-II, insulin, or glucagon. Levels of both IGFBPs in beta TC3 cell-conditioned media increased in the presence of dexamethasone at concentrations of 10(-6) M or greater. In summary, we present evidence that beta-cell lines comprise an environment for GH and IGF action. We speculate that IGFs, their receptors and binding proteins function as a complex interactive system which regulates beta-cell growth and function.

Leukotriene D4 induces MMP-1, which functions as an IGFBP protease in human airway smooth muscle cells.

We have previously demonstrated that the asthma-associated proinflammatory eicosanoid leukotriene D4 (LTD4) is comitogenic with insulin-like growth factors (IGF) in airway smooth muscle (ASM) cells. This synergistic effect of LTD4 and IGF on ASM cell growth involves proteolysis of ASM-produced inhibitory IGF-binding proteins (IGFBP). In this report, we analyzed the conditioned media (CM) from LTD4-treated human ASM cells (ASM-LTD4-CM) by Western ligand blotting and demonstrated a marked LTD4-induced reduction in the levels of the intact IGFBP (predominantly IGFBP-2) secreted by these cells. The IGFBP-2 in the ASM-LTD4-CM was identified as lower-molecular-weight fragments by Western immunoblotting. Incubation with 125I-labeled IGFBP demonstrated that an IGFBP protease was induced in the ASM cells in response to LTD4 treatment. Immunodepletion of ASM-LTD4-CM with anti-matrix metalloproteinase (MMP)-1 antibodies demonstrated a dose-dependent reduction of IGFBP proteolysis. Tissue inhibitor of MMP-1 and Batimastat (synthetic) inhibited proteolysis of IGFBP. Immunoblotting the ASM-LTD4-CM with anti-MMP-1 demonstrated a dose-dependent increase in MMP-1 protein. Similar results were also obtained by immunocytochemistry. Collectively, these observations demonstrate that MMP-1 is an IGFBP protease induced by leukotrienes that plays a significant role in modulating IGF action in ASM cells. A similar mechanism may be applicable in vivo in the airways of patients with asthma.

7S nerve growth factor is an insulin-like growth factor-binding protein protease.

Insulin-like growth factor (IGF)-binding protein (IGFBP) proteases modulate IGF action by cleaving IGFBPs into fragments with lower affinity to IGFs, thereby increasing the levels of free IGFs. We have previously documented that prostate-specific antigen (PSA), a serine protease of the kallikrein family, is an IGFBP-3 protease. In this study, we characterized the potential IGFBP proteolytic activity of nerve growth factor (NGF gamma-subunit), which shares high sequence homology with PSA. [125I]IGFBP-3 was cleaved by NGF (but not other kallikreins) at a 3-fold lower concentration than that of PSA, thus proving NGF to be a more potent IGFBP protease than PSA. NGF-generated, lower mol wt IGFBP-3 fragments, detected by immunoblotting and cross-linking to IGFs, had a lower affinity to IGFs than intact IGFBP-3. Unlike PSA, which cleaves primarily IGFBP-3 and -5, NGF also displayed potent proteolytic activity against IGFBP-4 and -6. These data suggest that NGF may be involved in the growth of cells by more than one mechanism. In addition to binding to its receptors, NGF is capable of cleaving IGFBPs and, thus, enhancing IGF action. This synergistic action between NGF and IGF may have important implications on cell growth, development, and repair in the brain and other tissues.

The effect of growth hormone treatment on the insulin-like growth factor axis in a child with nonislet cell tumor hypoglycemia.

We have previously described a case of tumor-associated hypoglycemia secondary to the production of high molecular weight insulin-line growth factor (IGF)-II in a child with congenital neuroblastoma. The child's hypoglycemia resolved with GH therapy and has continued to be well controlled for 1 yr. This represents one of the first cases of nonislet cell tumor hypoglycemia (NICTH) treated successfully with long-term exogenous GH. We now present an in-depth analysis of the IGF axis in this patient, before and after GH treatment. Although IGF-II levels at presentation were in the normal range, they were inappropriate for the patient's low GH state. Furthermore, the percentage of "big" IGF-II was elevated, as was the level of the IGF-IIE peptide, which is normally cleaved in the processing of the mature peptide. On the initial evaluation, GH levels failed to rise in response to hypoglycemia, IGF-I levels were low, IGF binding protein-3 (IGFBP-3) levels were suppressed, and IGFBP-2 levels were elevated. We have shown that baseline IGFBP-3 levels were low by RIA and immunoblotting and have demonstrated that this decrease was not associated with IGFBP protease activity. We have also demonstrated the baseline suppression of the acid labile subunit (ALS) of the 150K ternary complex by a novel immunoblot assay. The ratio of IGFs to IGFBP-3 was dramatically elevated, presumably leading to hypoglycemia. Furthermore, the percentage of serum IGF-I and IGF-II present as part of a binary (50K) complex with IGFBPs was also increased. GH therapy resulted in a normalization of the levels of blood sugars, IGFBP-3, ALS, IGFBP-2, and IGF-I, as well as the IGF/IGFBP-3 ratio. In summary, we have presented evidence that the hypoglycemia in this patient resulted from tumor production of high molecular weight IGF-II, which suppressed GH secretion, leading to the described derangements in the IGF binding proteins. We speculate that as a result of the decreased IGFBP-3 and ALS levels, the IGF population was shifted from the stable 150K complex to lower molecular weight complexes with IGF binding proteins, increasing IGF availability to tissues due to rapid turnover of these low molecular weight complexes. We demonstrated the reversal of the abnormalities in the IGFBP levels with GH treatment, corresponding to the clinical response of euglycemia.

Leukotriene D4 facilitates airway smooth muscle cell proliferation via modulation of the IGF axis.

The insulin-like growth factor (IGF) axis is involved in regulating proliferation in a variety of cell types, including airway smooth muscle. Because airway hyperplasia is a characteristic feature of asthma and other lung diseases, we examined the interaction of the potent proinflammatory eicosanoid leukotriene D4 (LTD4) with the IGF axis in regulating airway smooth muscle cell mitogenesis. In cultured rabbit airway smooth muscle cells, IGF-I but not LTD4 was mitogenic at submaximal concentrations. The combination of the two agents exerted a significant synergistic effect on airway smooth muscle cell mitogenesis. Analysis of airway smooth muscle cell conditioned medium by Western ligand blotting demonstrated a marked LTD4-induced reduction in the levels of the predominant IGF binding protein IGFBP-2, which is elaborated into the conditioned medium. The latter effect on IGFBP-2 release was not associated with a reduction in IGFBP-2 mRNA levels; however, LTD4-treated airway smooth muscle conditioned medium demonstrated the presence of a lower molecular weight form of IGFBP-2 by cross-linking to IGFs and specific proteolysis of radiolabeled IGFBP-2. IGFBP-2 was also noted to be associated with airway smooth muscle cell membranes, where it was protected from LTD4-induced proteolysis. Finally, exogenous administration of IGFBP-2 was found to inhibit the promitogenic effect of IGF-I in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)