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Breast cancer (BCa) bone metastases cause osteolytic bone lesions, which result from the interactions of metastatic BCa cells with osteoclasts and osteoblasts. Osteoclasts differentiate from myeloid lineage cells. To understand the cell-specific role of transforming growth factor beta (TGF-ß) in the myeloid lineage, in BCa bone metastases, MDA-MB-231 BCa cells were intra-tibially or intra-cardially injected into LysMCre/Tgfbr2floxE2/floxE2knockout (LysMCre/Tgfbr2KO) or Tgfbr2floxE2/floxE2mice. Metastatic bone lesion development was compared by analysis of both lesion number and area. We found that LysMCre/Tgfbr2knockout significantly decreased MDA-MB-231 bone lesion development in both the cardiac and tibial injection models. LysMCre/Tgfbr2knockout inhibited the tumor cell proliferation, angiogenesis and osteoclastogenesis of the metastatic bones. Cytokine array analysis showed that basic fibroblast growth factor (bFGF) was downregulated in MDA-MB-231-injected tibiae from the LysMCre/Tgfbr2KO group, and intravenous injection of the recombinant bFGF to LysMCre/Tgfbr2KO mice rescued the inhibited metastatic bone lesion development. The mechanism by which bFGF rescued the bone lesion development was by promotion of tumor cell proliferation through the downstream mitogen-activated protein kinase (MAPK)-extracellular signal–regulated kinase (ERK)-cFos pathway after binding to the FGF receptor 1 (FGFR1). Consistent with animal studies, we found that in human BCa bone metastatic tissues, TGF-ß type II receptor (TßRII) and p-Smad2 were expressed in osteoclasts and tumor cells, and were correlated with the expression of FGFR1. Our studies suggest that myeloid-specific TGF-ß signaling-mediated bFGF in the bone promotes BCa bone metastasis.

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors has been shown to regulate proliferation in several cell types. Although recent studies have demonstrated aberrant expression or activity of NF-kappaB in human breast cancer cell lines and tumors, little is known regarding the precise role of NF-kappaB in normal proliferation and development of the mammary epithelium. We investigated the function of NF-kappaB during murine early postnatal mammary gland development by observing the consequences of increased NF-kappaB activity in mouse mammary epithelium lacking the gene encoding IkappaBalpha, a major inhibitor of NF-kappaB. Mammary tissue containing epithelium from inhibitor kappaBalpha (IkappaBalpha)-deficient female donors was transplanted into the gland-free mammary stroma of wild-type mice, resulting in an increase in lateral ductal branching and pervasive intraductal hyperplasia. A two- to threefold increase in epithelial cell number was observed in IkappaBalpha-deficient epithelium compared with controls. Epithelial cell proliferation was strikingly increased in IkappaBalpha-deficient epithelium, and no alteration in apoptosis was detected. The extracellular matrix adjacent to IkappaBalpha-deficient epithelium was reduced. Consistent with in vivo data, a fourfold increase in epithelial branching was also observed in purified IkappaBalpha-deficient primary epithelial cells in three-dimensional culture. These data demonstrate that NF-kappaB positively regulates mammary epithelial proliferation, branching, and functions in maintenance of normal epithelial architecture during early postnatal development.

The function of many transmembrane molecules can be altered by cleavage and subsequent release of their ectodomains. We have investigated ectodomain cleavage of the cell-cell adhesion and signal-transducing molecule E-cadherin. The E-cadherin ectodomain is constitutively shed from the surface of MCF-7 and MDCKts.srcC12 cells in culture. Release of the 80 kDa soluble E-cadherin fragment is stimulated by phorbol-12-myristate-13-acetate and is inhibited by overexpression of the tissue inhibitor of metalloproteinases-2. The metalloproteinases matrilysin and stromelysin-1 both cleave E-cadherin at the cell surface and release sE-CAD into the medium. The soluble E-cadherin fragment thus released inhibits E-cadherin functions in a paracrine way, as indicated by induction of invasion into collagen type I and inhibition of E-cadherin-dependent cell aggregation. Our results, therefore, suggest a novel mechanism by which metalloproteinases can influence invasion.

The use of somatic cell hybrids has led to an increased understanding of the 'negative' regulation of cellular phenotype. Using somatic cell hybrids constructed between human breast cells that represent differing stages of malignancy but also display differing phenotypes from the same tissue, we present experimental results suggesting that luminal epithelial characteristics are controlled by repressive mechanisms. Fusion of HBL 100 cells, non-tumorigenic and characteristic of the basal cell lineage, with MCF-7 or MDA-MB-468 malignant breast cancer cells, characteristic of the luminal lineage, resulted in hybrid cells that displayed the phenotype of the HBL 100 cells. Using representational difference analysis, a panel of genes whose expression was repressed in the hybrid between HBL 100 and MDA-MB-468 was identified. This analysis revealed markers of luminal epithelial cells to be repressed, including Ep-CAM, cytokeratin 19 and E-cadherin. These markers were found to be coordinately re-expressed in variant hybrid cells indicating that the observed repression is reversible. Integrin (alpha)(v)(beta)(3) expression was found to be in mutual exclusivity to the luminal epithelial markers, thereby revealing a bidirectional 'switch' in the pattern of gene expression in this system. Finally, the expression of Ep-CAM was found to be lost in heterokaryons produced by fusion of HBL 100 and MCF-7 or MDA-MB-468 cells suggesting that the extinction of this gene in hybrid cells is the consequence of a trans-acting factor(s) synthesized by the HBL 100 cells. These data suggest that a number of markers of luminal cell differentiation in the mammary gland can be controlled through negative mechanisms and that such control of phenotype is highly coordinated.

To examine the role of matrilysin (MAT), an epithelial cell-specific matrix metalloproteinase, in the normal development and function of reproductive tissues, we generated transgenic animals that overexpress MAT in several reproductive organs. Three distinct forms of human MAT (wild-type, active, and inactive) were placed under the control of the murine mammary tumor virus promoter/enhancer. Although wild-type, active, and inactive forms of the human MAT protein could be produced in an in vitro culture system, mutations of the MAT cDNA significantly decreased the efficiency with which the MAT protein was produced in vivo. Therefore, animals carrying the wild-type MAT transgene that expressed high levels of human MAT in vivo were further examined. Mammary glands from female transgenic animals were morphologically normal throughout mammary development, but displayed an increased ability to produce beta-casein protein in virgin animals. In addition, beginning at approximately 8 mo of age, the testes of male transgenic animals became disorganized with apparent disintegration of interstitial tissue that normally surrounds the seminiferous tubules. The disruption of testis morphology was concurrent with the onset of infertility. These results suggest that overexpression of the matrix-degrading enzyme MAT alters the integrity of the extracellular matrix and thereby induces cellular differentiation and cellular destruction in a tissue-specific manner.

Various oncogenes or epidermal growth factor (EGF) induce transcription of a 1.9-kilobase RNA (transin RNA) in rat fibroblasts. The induction by EGF can be blocked by cycloheximide. Thus the response of the transin gene to EGF appears to require de novo protein synthesis. Transin RNA induction is specific to EGF, as neither insulin, platelet-derived growth factor, fibroblast growth factor, nor transforming growth factor beta could elicit the same response. However, transforming growth factor beta could block the EGF induction of transin RNA. Whereas the calcium ionophore A23187 and the tumor promoter TPA, either alone or administered together, did not increase transin RNA levels, TPA could synergise with a serum factor to effect such an increase. Dibutyryl cyclic AMP also induced transin RNA. Treatment of cells with the microfilament-disrupting agent cytochalasin B, but not the microtubule-disrupting agent colcemid, resulted in an increase in transin RNA levels, suggesting a role for the cytoskeleton in control of transin gene expression. The transin RNA does not contain repeated sequences and appears to be encoded by a single-copy gene. The protein sequence encoded by the last four exons of the transin gene shows some homology to two regions of the heme-binding protein hemopexin.

We previously reported that 125I-labeled epidermal growth factor is processed intracellularly to acidic macromolecules in Rat-1 fibroblasts. The present study defines the precursor-product relationship and localization of the processing steps to subcellular organelles by the use of a single isoelectric species of 125I-epidermal growth factor and Percoll gradient fractionation. The native pI 4.55 125I-epidermal growth factor was rapidly processed to a pI 4.2 species on or near the cell surface and in organelles corresponding to clathrin-coated vesicles, Golgi, and endoplasmic reticulum. This species was then processed to a pI 4.35 species in similar organelles. The pI 4.2 and 4.35 species were converted to a pI 4.0 species in dense, lysosome-like organelles. This species was ultimately degraded and exocytosed from the cell as low molecular weight products.

Upon internalization, epidermal growth factor (EGF) is proteolytically processed from its COOH terminus as it traverses intracellular vesicles and lysosomes. This report describes experiments which were conducted to determine whether lysosomotropic amines such as methylamine, which are known to inhibit degradation of EGF, are able to significantly inhibit the COOH-terminal processing of EGF, and whether disruption of EGF processing would negatively affect EGF-stimulated events such as DNA synthesis and induction of specific mRNA species. The results of these experiments indicated that, whereas methylamine treatment had no effect on EGF binding or internalization, vesicular translocation from endocytic vesicles to lysosomes was halted and processing of EGF was severely inhibited. The stimulation of DNA synthesis beginning 12 h after EGF exposure was also markedly inhibited by methylamine treatment. However, addition of methylamine alone produced a non-specific inhibition of DNA synthesis. The ability of EGF to induce specific transcription of the rat transin gene within 6 h of treatment was also not inhibited by methylamine treatment, but was actually increased in the presence of methylamine. These results suggest that at least some early transcriptionally regulated events induced by EGF do not require vesicular processing of EGF (or its receptor) and that the signal transduced by the binding of EGF to its receptor occurs in, or proximal to, the endocytic vesicles.

We have developed a system for studying the proteinase activity of a collagenase family member, transin. Cos cells transfected with a vector designed to direct synthesis of a secretable fusion protein between staphylococcal protein A and transin secrete a latent proteinase, activable by 4-aminophenylmercuric acetate, which binds to IgG-Sepharose. Treatment with 4-aminophenylmercuric acetate leads to cleavage of the fusion protein and elution of the active proteinase transin. Based on results obtained with this system we propose that transin comprises an N-terminal proteinase domain and an independent C-terminal hemopexin-like domain. The latter domain is not required for binding of inhibitors or for maintenance of transin in its inactive form. The sequence PRCGVPDV is present in the proenzyme forms of collagenase family proteinases just upstream from the N termini of the active enzymes. We show that mutations within this sequence lead to transin variants with a much increased tendency to undergo spontaneous activation. Finally, we show that mutations within a region of transin having sequence similarity to the zinc-binding site of bacterial metalloproteinases inactivate the proteinase activity of transin, lending support to the notion that this region represents part of transin's active site.

Stromelysin-1 (ST-1) is an extracellular matrix metalloproteinase whose expression is transcriptionally regulated by nerve growth factor (NGF) in the PC12 rat pheochromocytoma cell line. In this paper, we define sequences in the proximal ST-1 promoter that contain a novel NGF-responsive element(s). We show that this cis-acting promoter element can bind nuclear proteins from both untreated and NGF-treated PC12 cells in a specific and saturable manner and is sufficient to confer NGF-inducibility to a heterologous promoter. At least a portion of this NGF-responsive element lies within a 12-base pair region between positions -241 and -229 of the ST-1 promoter and bears no sequence homology to other known transcriptional elements. In contrast to what has been reported for fibroblasts, an AP1 site centered around position -68 does not seem to be involved in the growth factor regulation of ST-1 in PC12 cells. These results suggest that the NGF regulation of ST-1 gene expression involves different promoter elements, and possibly different transcription factors, from that described for ST-1 induction by other growth factors.

Transin RNA is a 1.9-kilobase RNA transcript induced by oncogenes in rat embryo fibroblast cell lines. We show that RNA species complementary to a cloned transin cDNA are present in mouse skin squamous cell carcinomas induced by a classical initiation-promotion protocol but not in premalignant, benign papillomas or in normal epidermis. A single application of a tumor promoting phorbol ester to normal epidermis elicits a transient increase in these RNA levels. Transin RNA encodes a secreted protease, an activity consistent with a functional role for enhanced expression of transin RNA in the progression of benign, encapsulated tumors to malignant, invasive carcinomas.

Transin mRNA encodes a secreted metalloprotease which is transcriptionally induced in Rat-1 cells by epidermal growth factor (EGF) and a number of oncogenes. A role for transin in tumor progression is suggested by its overexpression in malignant and metastatic tumors compared to their benign counterparts. In an effort to elucidate mechanisms by which elevated transin expression may be inhibited, it has been determined that both transforming growth factor type beta 1 (TGF beta 1) and increased levels of intracellular cyclic 5′-adenosine monophosphate (cAMP) inhibit EGF and oncogene induction of transin mRNA. The inhibition of transin mRNA occurred at the level of transcription as demonstrated by nuclear run-on assays. EGF binding studies in Rat-1 cells showed no significant effect of cAMP or TGF beta 1 on EGF receptor number or affinity. We have also examined the effects of cAMP and TGF beta 1 on oncogene-induced transin using Rat-1 cells transformed by temperature-sensitive mutants of v-src and K-ras oncogenes. Both inhibitors prevented the induction of transin RNA as well as decreased the levels of transin once elevated at the permissive temperature. Despite the similarities in the actions of TGF beta 1 and cAMP on transin gene expression, TGF beta 1 treatment did not significantly elevate intracellular cAMP levels, thus making it unlikely that cAMP is a second messenger system for TGF beta 1 action. These studies suggest that the inhibitory effects of cAMP and TGF beta 1 occur by distinct pathways at the level of gene regulation.

Cholecystokinin octapeptide (CCK8), the COOH-terminal moiety of cholecystokinin (CCK), exerted a rapid inhibitory effect on total cell-associated 125I-labeled epidermal growth factor (125I-EGF) binding by decreasing the rate of EGF internalization in isolated rat pancreatic acini. Removal of CCK8 from incubation medium followed by extensive washing of acini did not abolish its inhibitory effect, indicating that its action was not readily reversible. Proglumide, a competitive antagonist of CCK8, blocked the inhibitory action of the secretagogue. Addition of CCK8 to cells previously exposed to 125I-EGF did not enhance the release of cell-associated 125I activity. CCK8 did not inhibit the binding of 125I-labeled insulin to pancreatic acini. Other pancreatic secretagogues that enhance digestive-enzyme release through Ca2+, including caerulein, bombesin, carbachol, gastrin, and the Ca2+ ionophore A23187, also inhibited cell-associated 125I-EGF radioactivity. Further, at 37 degrees C the ionophore A23187 inhibited specific 125I-EGF binding in human A-431 carcinoma cells, Swiss 3T3 cells, and Rat-1 fibroblasts, and this effect was abolished when 125I-EGF internalization was reduced by incubating cells at 4 degrees C. It is concluded that alterations in cellular Ca2+ in the pancreas and other cells lead to inhibition of EGF endocytosis.