Matrix metalloproteinase 20-dentin sialophosphoprotein interaction in oral cancer.

Matrix metalloproteinase 20 (MMP-20), widely regarded as tooth specific, participates with MMP-2 in processing dentin sialophosphoprotein (DSPP) into dentin sialoprotein, dentin phosphoprotein, and dentin glycoprotein. In biochemical system, MMP-2, MMP-3, and MMP-9 bind with high affinity to, and are activated by, specific small integrin-binding ligand N-linked glycoproteins (SIBLINGs): bone sialoprotein, osteopontin, and dentin matrix protein 1, respectively. Subsequent reports documented possible biological relevance of SIBLING-MMP interaction in vivo by showing that SIBLINGs are always coexpressed with their MMP partners. However, the cognate MMPs for 2 other SIBLINGs-DSPP and matrix extracellular phosphogylcoprotein-are yet to be identified. Our goal was to investigate MMP-20 expression and to explore preliminary evidence of its interaction with DSPP in oral squamous cell carcinomas (OSCCs). Immunohistochemistry analysis of sections from 21 cases of archived human OSCC tissues showed immunoreactivity for MMP-20 in 18 (86%) and coexpression with DSPP in all 15 cases (71%) positive for DSPP. Similarly, 28 (93%) of 30 cases of oral epithelial dysplasia were positive for MMP-20. Western blot and quantitative real-time polymerase chain reaction analysis on OSCC cell lines showed upregulation of MMP-20 protein and mRNA, respectively, while immunofluorescence showed coexpression of MMP-20 and DSPP. Colocalization and potential interaction of MMP-20 with dentin sialoprotein was confirmed by coimmunoprecipitation and mass spectrometry analysis of immunoprecipitation product from OSCC cell lysate, and in situ proximity ligation assays. Significantly, results of chromatin immunoprecipation revealed a 9-fold enrichment of DSPP at MMP-20 promoter-proximal elements. Our data provide evidence that MMP-20 has a wider tissue distribution than previously acknowledged. MMP-20-DSPP specific interaction, excluding other MMP-20-SIBLING pairings, identifies MMP-20 as DSPP cognate MMP. Furthermore, the strong DSPP enrichment at the MMP-20 promoter suggests a regulatory role in MMP-20 transcription. These novel findings provide the foundation to explore the mechanisms and significance of DSPP-MMP-20 interaction in oral carcinogenesis.

Upregulation of alveolar levels of activin B, but not activin A, in lungs of west highland white terriers with idiopathic pulmonary fibrosis and diffuse alveolar damage.

Activins, cytokines belonging to the transforming growth factor-ß superfamily, have an important role in inflammation and fibrosis. Activin A has been suggested to participate in the pathophysiology of human idiopathic pulmonary fibrosis (IPF), but studies on the role of activin B are sparse. Canine IPF (CIPF) is an incurable interstitial lung disease occurring particularly in West Highland white terriers (WHWTs). During the disease course there are acute exacerbations (AEs) and the condition has a poor prognosis. Microscopically, AEs of CIPF are characterized by diffuse alveolar damage, which is also a key feature of acute respiratory distress syndrome (ARDS). The aim of this study was to study expression of activin A and B in lung tissue of WHWTs with CIPF and WHWTs with CIPF and concurrent AE, and dogs of various breeds with ARDS and to compare these findings with those of healthy WHWTs. In addition, western blot analysis of activin B from bronchoalveolar lavage fluid (BALF) from WHWTs with CIPF and healthy WHWTs was conducted. Activin B, but not activin A, was strongly expressed in the altered alveolar epithelium in the lungs of WHWTs with CIPF as well as in the lungs of dogs with ARDS. Activin B was detected in the BALF of WHWTs with CIPF, most notably in samples from dogs with AE, but activin B was not detected in BALF from healthy WHWTs. These findings suggest that activin B may be part of the pathophysiology of CIPF and might act as a marker of alveolar epithelial damage.

Comparative study of transforming growth factor-ß signalling and regulatory molecules in human and canine idiopathic pulmonary fibrosis.

Activation of transforming growth factor (TGF)-ß is a key event in the progression of fibrosis in human lung tissue. Idiopathic pulmonary fibrosis (IPF) in West Highland white terriers (WHWTs) shares histopathological features of human usual interstitial pneumonia (UIP), the histopathological counterpart of IPF and non-specific interstitial pneumonia (NSIP). The aim of the present immunohistochemical study was to investigate TGF-ß signalling activity and its known extracellular matrix (ECM) regulatory proteins, latent TGF-ß binding protein (LTBP)-1 and fibrillin-2, in lung tissue of WHWTs with IPF and healthy WHWTs and to compare these with findings in human UIP and NSIP. P-Smad2 immunoreactivity, indicating TGF-ß signalling activity, was increased in WHWTs with IPF relative to healthy WHWTs and expression was localized predominantly in the altered alveolar epithelium, as seen in both UIP and NSIP. Increased peribronchial and perivascular LTBP-1 immunoreactivity was seen in WHWTs with IPF compared with controls, possibly indicating the importance of the small airways in the canine disease. Alveolar LTPB-1 immunolabelling in diseased WHWTs was seen mainly in the altered alveolar epithelium, resembling more closely the labelling in UIP than in NSIP. Alveolar interstitial fibrillin-2 immunoreactivity, which is up-regulated in the lungs of people with UIP, was also detected in the lungs of WHWTs with IPF and people with NSIP. However, no significant difference was seen between WHWTs with IPF and control WHWTs. The results suggest that increased TGF-ß signalling and expression of the ECM regulatory proteins LTBP-1 and fibrillin-2 are part of the molecular pathophysiology of canine IPF.

Gremlin-1 associates with fibrillin microfibrils in vivo and regulates mesothelioma cell survival through transcription factor slug.

Malignant mesothelioma is a form of cancer that is highly resistant to conventional cancer therapy for which no major therapeutic advances have been introduced. Here, we identify gremlin-1, a known bone morphogenetic protein inhibitor crucial for embryonic development, as a potential therapeutic target for mesothelioma. We found high expression levels of gremlin-1 in the mesothelioma tumor tissue, as well as in primary mesothelioma cells cultured from pleural effusion samples. Downregulation of gremlin-1 expression by siRNA-mediated silencing in a mesothelioma cell line inhibited cell proliferation. This was associated with downregulation of the transcription factor slug as well as mesenchymal proteins linked to cancer epithelial-to-mesenchymal transition. Further, resistance to paclitaxel-induced cell death was associated with high gremlin-1 and slug expression. Treatment of gremlin-1-silenced mesothelioma cells with paclitaxel or pemetrexed resulted in efficient loss of cell survival. Finally, our data suggest that concomitant upregulation of fibrillin-2 in mesothelioma provides a mechanism for extracellular localization of gremlin-1 to the tumor microenvironment. This was supported by the demonstration of interactions between gremlin-1, and fibrillin-1 and -2 peptides as well as by colocalization of gremlin-1 to fibrillin microfibrils in cells and tumor tissue samples. Our data suggest that gremlin-1 is also a potential target for overcoming drug resistance in mesothelioma.

Gremlin localization and expression levels partially differentiate idiopathic interstitial pneumonia severity and subtype.

Idiopathic pulmonary fibrosis (IPF) (histopathology of usual interstitial pneumonia, UIP) and non-specific interstitial pneumonia (NSIP) are diseases characterized by loss of normal lung architecture and function. The differential diagnosis between IPF/UIP and NSIP may be difficult. The levels of bone morphogenetic protein (BMP)-4 antagonist gremlin are up-regulated in IPF/UIP. The present study was performed to clarify whether the localization or the mRNA expression of gremlin or BMP-4 could be used in the differential diagnosis or assessment of severity of IPF/UIP and NSIP. Gremlin and BMP-4 immunoreactivities were quantitated from 24 UIP and 12 NSIP lung specimens. Quantitative real-time polymerase chain reaction analyses were performed to compare gremlin and BMP-4 expression between UIP (n = 8) and NSIP (n = 5) biopsies. Immunohistochemical positivity and mRNA levels were correlated to lung function parameters. In IPF/UIP biopsies, gremlin was detected mainly in the thickened lung parenchyma, whereas in NSIP it was observed in the alveolar epithelium. BMP-4-positive (BMP-4+) cells were detected solely in the alveolar wall. The percentage of gremlin-positive area was higher in IPF/UIP (5.1 +/- 0.6) than in NSIP (1.8 +/- 0.7) (n = 36, p < 0.0001). Gremlin mRNA levels were higher in advanced UIP (p = 0.008) and NSIP (p = 0.007) biopsies than in the normal control lung. A negative correlation was found between the specific diffusion capacity corrected for alveolar volume (DLCO/VA) and gremlin mRNA levels (r = - 0.69, p = 0.007). The highest numbers of BMP-4+ cells were found in NSIP biopsies. BMP-4 mRNA levels correlated positively with forced vital capacity (r = 0.801, p < 0.0001) and diffusion capacity. Parenchymal gremlin immunoreactivity is thus suggestive of a UIP-type interstitial pneumonia. Gremlin expression levels correlating negatively and BMP-4 levels positively with disease severity support recent observations of a fibroprotective role for the BMPs.

Activation of Smad signaling enhances collagenase-3 (MMP-13) expression and invasion of head and neck squamous carcinoma cells.

Squamous cell carcinoma (SCC) cells of the head and neck specifically express collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the expression of which correlates with their invasion capacity. Transforming growth factor-beta (TGF-beta) enhances MMP-13 and collagenase-1 (MMP-1) expression and invasion of SCC cells via p38 mitogen-activated protein kinase. Here, we have examined the role of Smad signaling in regulating MMP-13 expression and in invasion of head and neck SCC cells. Treatment with TGF-beta resulted in activation of Smad2 and Smad3 in SCC cells, but had no effect on their proliferation or viability. Basal activation of Smad3 and p38 was noted in SCC cells without exogenous TGF-beta stimulation, and adenoviral delivery of Smad7 and dominant-negative Smad3 inhibited p38 activation in these cells. Adenoviral overexpression of Smad3 augmented the upregulatory effect of TGF-beta on MMP-13 expression by SCC cells. Disruption of Smad signaling by adenoviral expression of kinase-defective TGF-beta type I receptor (activin-receptor-like kinase-5), Smad7, and dominant-negative Smad3 potently suppressed the basal and TGF-beta-induced expression of MMP-13 and MMP-1 in SCC cells, and inhibited their basal and TGF-beta-induced invasion through Matrigel and type I collagen. Adenoviral overexpression of Smad7 in cutaneous and oral SCC cells significantly inhibited their implantation in skin of SCID mice and growth of xenografts in vivo, as compared to LacZ adenovirus-transduced control cells. Together, these results show that Smad signaling plays an important role in promoting the invasive phenotype of human head and neck SCC cells by upregulating their collagenase expression.

Novel non-TGF-beta-binding splice variant of LTBP-4 in human cells and tissues provides means to decrease TGF-beta deposition.

Small latent TGF-beta consists of latency associated peptide (LAP) bound to the 25 kDa TGF-beta by noncovalent interactions. Small latent TGF-beta is secreted from cells and deposited into the extracellular matrix as covalent complexes with its binding proteins, LTBPs. Four LTBPs have been molecularly cloned and their structures contain repetitive sequences. The 3rd 8-Cys repeats of LTBP-1, -3 and -4 are able to associate with small latent TGF-beta. We analyzed by RT-PCR the expression of LTBPs 1-4 in a panel of cultured human cell lines including fibroblasts of different origin, endothelial cells and immortalized keratinocytes. LTBPs were expressed in an overlapping manner, but differences in their expression levels were detected. SV-40 transformed human embryonic lung fibroblasts contained less of the mRNAs for the LTBPs, suggesting that malignant transformation leads to decrease in LTBP expression. A novel alternatively spliced form of LTBP-4 lacking the 3rd 8-Cys repeat (LTBP-4delta8-Cys3rd) was identified. LTBP-4delta8-Cys3rd does not bind TGF-beta and it was found to be expressed in the same tissues as the full length LTBP-4. The exon-intron structure of LTBP-4 around the 3rd 8-Cys repeat was similar to those of LTBP-2 and -3. LTBP-4delta8-Cys3rd was produced by alternative splicing over two exons. In addition, HL-60 promyelocytic leukemia cells expressed a splice variant lacking only one exon of this region. The expression of the non-TGF-beta-binding variant of LTBP-4 may be important for the regulation of TGF-beta deposition in tissues. Since LTBPs are a part of the extracellular matrix microfibrils, the LTBP-4delta8-Cys3rd protein may also be involved in various structural functions not related to TGF-beta signaling.

Latency, activation, and binding proteins of TGF-beta.

The TGF-beta superfamily of growth factors consists of an increasing number of different polypeptide modulators of cell growth, differentiation, and morphogenesis. Three mammalian isoforms have been molecularly cloned. Numerous ways to regulate the expression of the TGF-beta genes have been identified. TGF-betas are, for example, subject to regulation by retinoids, steroid hormones, and vitamin D. A characteristic feature in the biology of TGF-betas is that they are usually secreted from cells in latent forms. The large latent complex consists of the small latent complex (TGF-beta and its propeptide) and a high molecular weight protease resistant binding protein, latent TGF-beta binding protein (LTBP). LTBPs are required for the proper folding and secretion of TGF-beta. TGF-beta is not just secreted from cultured cells but is deposited via LTBPs to the pericellular space, namely to the extracellular matrix. Release of these complexes and activation by proteases is under tight regulation and provides a means to rapidly increase local concentrations of TGF-beta. Biological events, where enhanced or focal proteolysis and activation of latent TGF-beta takes place, include cell invasion, tissue remodeling, and wound healing.

1alpha,25-dihydroxyvitamin D3 and its analogues down-regulate cell invasion-associated proteases in cultured malignant cells.

Vitamin D and its derivatives (deltanoids) are potent regulators of cell proliferation and differentiation. Targeted production of proteolytic enzymes like serine proteases and metalloproteinases is an important part of the invasive process of cancer cells. Treatment with 1 alpha25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] decreases the invasive properties of breast carcinoma cells. Here we have analyzed the effects of 1alpha,25(OH)2D3 and its synthetic analogues on the secretion and cell surface association of the components of the plasminogen activator (PA) system and on the secretion of certain matrix metalloproteinases (MMPs) and their inhibitors in MDA-MB-231 breast carcinoma cells. Deltanoids were able to decrease the secretion of urokinase PA and tissue-type PA activity in a dose-dependent manner and to increase PA inhibitor 1 secretion, leading to reduced total PA activity. CB1093 was the most potent analogue, effective at concentrations several logarithms lower than 1alpha,25(OH)2D3. Transient transfection of different urokinase PA promoter reporter constructs to HT-1080 fibrosarcoma indicator cells indicated that vitamin D-responsive sequences were located between nucleotides -2350 and -1870 in the 5' region of the promoter. Treatment of MDA-MB-231 cells with 1alpha,25(OH)2D3 or other deltanoids also resulted in decreased MMP-9 levels in association with increased tissue inhibitor of MMP 1 activity. Membrane-type 1-MMP expression or proteolytic processing were not appreciably affected by deltanoids. Vitamin D and its analogues caused a decrease in Matrigel invasion assays of MDA-MB-231 cells. Cancer cell invasion is associated with coordinated secretion of proteolytic enzymes and their inhibitors. Vitamin D and its derivatives can evidently influence invasive processes by two means: (a) decreasing the expression and activity of cell invasion-associated serine proteases and metalloproteinases; and (b) inducing their inhibitors.

Reversal of tamoxifen resistance of human breast carcinomas in vivo by neutralizing antibodies to transforming growth factor-beta.

BACKGROUND: Overexpression of transforming growth factor (TGF)-beta has been reported in human breast carcinomas resistant to antiestrogen tamoxifen, but the role of TGF-beta in this resistant phenotype is unclear. We investigated whether inhibition of TGF-beta2, which is overexpressed in LCC2 tamoxifen-resistant human breast cancer cells, could modify antiestrogen resistance. METHODS: TGF-beta2 expression was evaluated in LCC2 cells and tamoxifen-sensitive LCC1 cells by northern blot analysis. Secreted TGF-beta activity was quantified by use of an 125I-TGF-beta competitive radioreceptor assay. Sensitivity to tamoxifen was measured in a soft agarose colony-forming assay and in a xenograft model in nude and beige/nude mice. Natural killer (NK) cell cytotoxicity was measured by 51Cr release from LCC1 and LCC2 cell targets coincubated with human peripheral blood mononuclear cells. Decrease in TGF-beta2 expression in LCC2 cells was achieved by treatment with antisense oligodeoxynucleotides and confirmed by TGF-beta2 immunoblot analysis. RESULTS AND CONCLUSIONS: The proliferative response of LCC2 cells to tamoxifen in vitro was not altered by TGF-beta neutralizing antibodies. However, established LCC2 tumors in nude mice treated with tamoxifen plus TGF-beta antibodies failed to grow, whereas tumors treated with tamoxifen plus a control antibody continued to proliferate. This reversal of tamoxifen resistance by TGF-beta antibodies did not occur in beige/nude mice, which lack NK-cell function, suggesting that immune mechanisms may be involved in the antitumor effects of tamoxifen. Antisense TGF-beta2 oligodeoxynucleotides enhanced the NK sensitivity of LCC2 cells in the presence of tamoxifen. Finally, LCC1 tumors were markedly more sensitive to tamoxifen in NK-active than in NK-deficient mice. IMPLICATIONS: These data suggest that host NK function mediates, in part, the antitumor effect of tamoxifen and that TGF-beta2 may abrogate this mechanism, thus contributing to tamoxifen resistance.

A kinase-defective transforming growth factor-beta receptor type II is a dominant-negative regulator for human breast carcinoma MCF-7 cells.

The role of transforming growth factor (TGF)-beta type II receptor (T beta RII) in TGF-beta resistance and tumor progression is now well recognized. To test the effects of T beta RII loss in determining malignancy, we transfected a T beta RII-expressing, TGF-beta-sensitive, MCF-7 cell strain (ME24) with a tetracycline-repressible truncated T beta RII (kdT beta RII) construct lacking the cytoplasmic domain of the receptor. Transfection of kdT beta RII into parental ME24 cells (designated ME24t6 after transfection) resulted in high expression levels of kdT beta RII mRNA and cell surface protein which were reversible by tetracycline treatment. ME24t6 cells did not respond to exogenous TGF-beta 1 as measured by inhibition of proliferation or fibronectin (FN) induction, indicating that the truncated T beta RII acted as a dominant-negative inhibitor of both the growth inhibitory and extracellular matrix (ECM) stimulatory TGF-beta effects. Furthermore, inhibition of kdT beta RII expression by tetracycline treatment led to TGF-beta 1-mediated cell growth arrest in the G1 phase of cell cycle and to the accumulation of the hypophosphorylated form of retinoblastoma (Rb) protein. However, compared to parental ME24 cells, transfectants failed to show increased tumorigenicity, indicating that loss of T beta RII itself is not sufficient to account for differences in the malignant properties of T beta RII-expressing and non-expressing MCF-7 cell strains.

Blockade of transforming growth factor-beta signaling does not abrogate antiestrogen-induced growth inhibition of human breast carcinoma cells.

We have studied the role of autocrine transforming growth factor-beta (TGF-beta) signaling on antiestrogen-mediated growth inhibition of hormone-dependent T47D and MCF-7 human breast carcinoma cells. Tamoxifen treatment increased the secretion of TGF-beta activity into serum-free cell medium and the cellular content of affinity cross-linked type I and III TGF-beta receptors in both cell lines. Anti-pan-TGF-beta antibodies did not block anti-estrogen-induced recruitment in G1 and inhibition of anchorage-dependent and -independent growth of both cell lines. Early passage MCF-7 cells, which exhibit detectable type II TGF-beta receptors at the cell surface and exquisite sensitivity to exogenous TGF-beta1, were transfected with a tetracycline-controllable dominant-negative TGF-betaRII (DeltaRII) construct. Although the TGF-beta1 response was blocked by removal of tetracycline in MCF-7/DeltaRII cells, tamoxifen-mediated suppression of Rb phosphorylation, recruitment in G1, and inhibition of cell proliferation were identical in the presence and absence of tetracycline. TGF-beta1 treatment up-regulated the Cdk inhibitor p21 and induced its association with Cdk2 in MCF-7 cells; these responses were blocked by the DeltaRII transgene product. In MCF-7 cells with a functional TGF-beta signaling pathway, tamoxifen did not up-regulate p21 nor did it induce association of p21 with Cdk2, suggesting alternative mechanisms for antiestrogen-mediated cytostasis. Finally, transfection of late-passage, TGF-beta1 unresponsive MCF-7 cells with high levels of TGF-betaRII restored TGF-beta1-induced growth inhibition but did not enhance tamoxifen response in culture. Taken together these data strongly argue against any role for TGF-beta signaling on tamoxifen-mediated growth inhibition of hormone-dependent breast cancer cells.

Processing of the transforming growth factor beta type I and II receptors. Biosynthesis and ligand-induced regulation.

Three cell surface transforming growth factor beta (TGFbeta) receptor (R) proteins regulate the effects of TGFbeta isoforms on growth and differentiation. TGFbeta-IR and -IIR are transmembrane serine/threonine kinases directly mediating the signaling across the plasma membrane. Both TGFbeta and its receptors are ubiquitously expressed, hence the fine regulation of the multiplicity of responses most likely involves several levels of control including the regulation of expression, complex formation, and down-regulation of the receptor proteins. In mink lung epithelial cells, TGFbeta-IIR was first synthesized as a approximately 60-kDa endoglycosidase H-sensitive precursor protein, which was converted to a mature approximately 70-kDa protein. The half-life of metabolically labeled mature TGFbeta-IIR was estimated to be 60 min and was further reduced to approximately 45 min in the presence of exogenous TGFbeta1. Minimal internalization of 125I-TGFbeta1 at 37 degrees C was detected suggesting that the rapid turnover was not due to endocytosis and degradation of the ligand-receptor complexes. TGFbeta-IR was synthesized as a approximately 53-kDa precursor protein, which was processed to a mature approximately 55-kDa receptor protein. The half-life of TGFbeta-IR was >12 h. A fraction of tunicamycin-treated type I and II receptors that reach the cell surface was able to associate in the presence of ligand suggesting that heteromeric complexes can form in a post-endoplasmic reticulum compartment before full glycosylation is achieved. These results show differential processing and turnover of TGFbeta-IR and TGFbeta-IIR providing a potential additional mechanism for modulation of cellular responses to TGFbetas.

Predominant cytosolic localization of type II transforming growth factor beta receptors in human breast carcinoma cells.

It is proposed that loss of a growth-inhibitory response to transforming growth factor beta (TGFbeta) contributes to breast cancer progression. Because cellular TGFbeta responsiveness often correlates with TGFbeta type II receptor (TGFbeta-IIR) expression, we have examined the cellular distribution of TGFbeta-IIRs in tumor and nontumor mammary epithelial cells. By immunoblot analysis, TGFbeta-IIR was detected both in membrane and cytosolic fractions of MDA-231 tumor cells as well as in normal human breast epithelial cells. The cytosolic protein appeared to be more abundant and was detected as a clear perinuclear staining by immunocytochemistry. The glycosylation patterns of the cytosolic and membrane form were different, indicating distinct receptor pools. The cytosolic TGFbeta-IIR did not bind 125I-labeled TGFbeta1 but had a detectable in vitro and in vivo kinase activity. MCF-7 breast cancer cells express the TGFbeta-IIR mRNA but show undetectable cell surface TGFbeta-IIR protein by affinity cross-linking. However, low levels of TGFbeta-IIR were observed in MCF-7 cytosol. Sequencing of the coding region of TGFbeta-IIR from MCF-7 cells indicated a point mutation (A439V) in a nonconserved region of the kinase domain. When MCF-7 cells were treated with sublethal doses of Adriamycin that induce cell differentiation, the membrane localization of TGFbeta-IIR and TGFbeta response were restored. Our results indicate the presence of a prominent, kinase-active TGFbeta-IIR in the cytosol of several mammary cell lines. This cytosolic pool of receptors is the only detectable one in MCF-7 cells. Loss of wild-type membrane receptors due to defects in trafficking presents a potential new mechanism for escape from negative growth control.

Complex role of tumor cell transforming growth factor (TGF)-beta s on breast carcinoma progression.

Growth inhibition by the TGF-beta s has been extensively studied in both normal and transformed mammary epithelial cells. It has been proposed that loss of autocrine TGF-beta mediated growth regulation is a critical event in breast tumorigenesis and several lines of in vitro and in vivo data support this hypothesis. However, a positive association between the expression of TGF-beta s by tumor cells and the progression or maintenance of breast cancinoma cells has been observed in many studies in in vivo tumor models. Possible mechanisms for these growth enhancing effects of TGF-beta include immunosuppression mediated by tumor TGF-beta s, enhanced angiogenesis, increased peritumoral stroma formation, and cell adhesion. The net effect of tumor cell TGF-beta on the biology of breast carcinogenesis would depend on the balance between autocrine growth inhibition of mammary epithelial cells and these growth enhancing effects.

Vitamin D3 regulation of transforming growth factor-beta system in epithelial and fibroblastic cells--relationships to plasminogen activation.

Vitamin D3 and its analogs are potent regulators of growth and differentiation of various cell types. A mechanism of action of vitamin D3 and other steroid hormones is to enhance the secretion of transforming growth factor-beta (TGF-beta) in target cells. In epidermal keratinocytes, vitamin D3 induced the expression of both TGF-beta 1 and TGF-beta 2 with minor changes in mRNA levels, while in BT-20 breast carcinoma cells the increase in TGF-beta activity was preceded by an induction of mRNA. In both cell systems, the absolute amounts of active TGF-beta increased, and in keratinocytes the proportion of active TGF-beta was also enhanced. A concomitant enhancement of secretion of the latent TGF-beta-binding protein by vitamin D3 was observed in BT-20 cells. Retinoic acid, which is known to interfere with vitamin D3 signaling, slightly decreased the levels of secreted TGF-beta 1 protein in BT-20 cells, but did not significantly affect the vitamin D3-induced increase. In addition to regulation of the TGF-beta system, vitamin D3 decreases pericellular plasminogen activator activity in keratinocytes. Plasmin-mediated proteolytic events are involved in the release from pericellular space and activation of TGF-beta. We analyzed vitamin D3 regulation of fibroblast growth and the secretion of PA activity. Vitamin D3 inhibited fibroblast growth in a concentration-dependent manner and downregulated plasminogen activator activity as in keratinocytes. In fibroblasts, vitamin D3 did not induce notable alterations in TGF-beta 1 or latent TGF-beta-binding protein secretion, suggesting divergent growth inhibitory mechanisms. Our results indicate that vitamin D3 and its analogs are potent regulators of the TGF-beta and plasminogen activator systems in cells of epithelial and mesenchymal origin.

Transforming growth factor-beta system and its regulation by members of the steroid-thyroid hormone superfamily.

TGF-beta s and their receptors are expressed ubiquitously, and they act as key regulators of many aspects of cell growth, differentiation, and function. Steroid action on target tissues is often associated with increase in TGF-beta isoforms. Regulation of TGF-beta expression and activation is crucial for normal development and growth control. The loss of responsiveness of different tumor cells to the antiproliferative effects of TGF-beta is a common feature in carcinogenesis. Multiple changes are required for the cells to gain complete resistance to TGF-beta growth inhibition (Fynan and Reiss, 1993; Kimchi et al., 1988; Samuel et al., 1992). Although many tumor cells are not growth inhibited by TGF-beta, they respond to TGF-beta treatment by changes in the expression of matrix components and enhanced proteolytic activity (KeskiOja et al., 1988). Agents that induce TGF-beta production in target tissues can have a chemopreventive or chemotherapeutic value for the management of epithelial malignancies. Conversely, data supporting a positive role for TGF-beta in established tumor progression are beginning to emerge (Arteaga et al., 1993a,b; Barrett-Lee et al., 1990; Arrick et al., 1992 ; E. A. Thompson et al., 1991). In later stages of tumor development, cell proliferation is often not inhibited by TGF-beta, and tumor cells secrete large amounts of this growth factor (Fynan and Reiss, 1993). In vivo TGF-beta secreted by tumor or stromal cells can influence host responses such as a natural killer cell function and thus indirctly support tumor cell viability (Arteaga et al., 1993b). TGF-beta may also affect tumor growth indirectly by stromal effects and promotion of angiogenesis. TGF-beta may also be involved in the progression of breast tumors from the steroid-sensitive to steroid-insensitive state (King et al., 1989). Understanding of the net effect of TGF-beta in different stages of tumor development is critical for the evaluation of its therapeutic value in cancer treatment.

1,25-Dihydroxyvitamin D3 enhances the expression of transforming growth factor beta 1 and its latent form binding protein in cultured breast carcinoma cells.

Transforming growth factor beta s (TGF-beta s) are a family of polypeptide growth factors that regulate cellular growth, phenotype, and differentiation. TGF-beta s are synthesized as latent high molecular weight complexes that include the NH2-terminal remnant of the TGF-beta precursor (latency-associated protein) and, frequently, latent TGF-beta binding protein. After activation, TGF-beta s act as local mediators of hormonal responses in target tissues. TGF-beta functions as a negative growth regulator for both breast cancer cells and normal mammary epithelial cells. Vitamin D3 is growth inhibitory for the estrogen receptor-negative breast cancer cell line BT-20 and regulates TGF-beta expression in cultured keratinocytes. We studied here the effects of vitamin D3 and its analogues on TGF-beta expression and activity in BT-20 cells. It was found that vitamin D3 enhanced both TGF-beta 1 mRNA and secretion of the protein in a time- and dose-dependent manner. Analyses of the vitamin D3 responses in the presence of cycloheximide or actinomycin D indicated that the TGF-beta 1 mRNA induction was dependent on both protein and RNA synthesis. The amounts of latent TGF-beta binding protein were also increased in the conditioned medium but not in the pericellular matrix of vitamin D3-treated cultures. The amounts of active TGF-beta were enhanced in vitamin D3-treated cultures as well, suggesting autocrine or paracrine functions for the secreted growth factor. Some analogues of vitamin D3 (EB 1089, MC 903, and KH 1060) that are known to be potent inhibitors of breast cancer cell growth both in vitro and in vivo had similar or more pronounced inducing effects on TGF-beta 1 mRNA levels. The present results indicate that vitamin D3 and its analogues are potent inducers of both active and latent forms of TGF-beta 1 in BT-20 breast carcinoma cells and provide evidence for coordinated regulation of latent TGF-beta binding protein and TGF-beta 1.

Vitamin D3 and calcipotriol decrease extracellular plasminogen activator activity in cultured keratinocytes.

Vitamin D3, 1 alpha,25(OH)2D3, and its metabolites regulate the growth and differentiation of several cell types. Vitamin D3 and its analogue, calcipotriol (MC 903), inhibit the proliferation of cultured human and mouse keratinocytes and induce keratinocyte differentiation. Calcipotriol is effective in the treatment of psoriasis in which increased plasminogen activator activity has been reported. We analyzed therefore the effects of calcipotriol and vitamin D3 on the production of plasminogen activator (PA) activity in human keratinocytes and a mouse keratinocyte cell line. Caseinolysis-in-agarose assays indicated that vitamin D3 decreases total PA activity in both keratinocyte culture systems. Zymographic analyses of the medium indicated that the secreted activator was of the urokinase type (u-PA). A decrease was observed also in extracellular matrix and membrane-associated u-PA activity of vitamin D3 and calcipotriol treated cells. Immunoblotting analysis of the conditioned medium from human keratinocytes revealed a decrease in the u-PA protein levels. Accordingly, Northern hybridization analysis of the respective mRNAs indicated a rapid decrease in urokinase mRNA levels. Calcipotriol decreased u-PA activity also in the presence of inducers of u-PA activity like transforming growth factor-beta, epidermal growth factor, and phorbol-12-myristate-13-acetate. Calcipotriol also caused a decrease in tissue type PA (t-PA) activity of the keratinocytes. Most t-PA activity was associated with the extracellular matrices and cell membranes as revealed by zymographic analysis. Paradoxically, the secretion and deposition of the matrix of plasminogen activator inhibitor type 1 decreased in calcipotriol-treated cells. The results indicate that a major effect of vitamin D3 on cultured keratinocytes is a decrease of plasminogen activator activity.

Vitamin D3 and calcipotriol enhance the secretion of transforming growth factor-beta 1 and -beta 2 in cultured murine keratinocytes.

Vitamin D3 and its analogue calcipotriol (MC 903) inhibit the proliferation of cultured keratinocytes and induce their differentiation. Since TGF beta s are very potent inhibitors of keratinocyte growth we studied the effects of vitamin D3 and calcipotriol on the secretion of TGF beta in cultured murine keratinocytes. Vitamin D3 and calcipotriol (10(-6)-10(-9) M) inhibited the DNA-synthesis of mouse keratinocytes by 50-80% in a time and dose-dependent manner as measured by [3H]-thymidine incorporation. Analysis of the conditioned medium of the keratinocytes indicated that the cells secreted into their medium activity that inhibited the growth of indicator Mv1Lu mink lung epithelial cells. Neutralizing antibodies against TGF beta 1 and TGF beta 2 decreased, and when used together, prevented the observed growth inhibition of the indicator cells. Heat treatment of the conditioned medium, which activates latent forms of TGF beta, revealed higher levels of growth inhibitory activity in the medium from vitamin D3 and calcipotriol treated than from control cultures indicating that a fraction of TGF beta was in a latent form. Active TGF beta was, however, detected considerably more in vitamin D3 and calcipotriol treated cultures than in control cultures. Immunoblotting analysis of the medium revealed enhanced secretion of TGF beta protein. These results indicate that enhanced TGF beta 1 and TGF beta 2 secretion and activity is associated with vitamin D3-mediated growth inhibition of cultured keratinocytes.