Characterisation of the immune microenvironment of primary breast cancer and brain metastasis reveals depleted T-cell response associated to ARG2 expression.

BACKGROUND: Immune checkpoint inhibition is an established treatment in programmed death-ligand 1 (PD-L1)-positive metastatic triple-negative (TN) breast cancer (BC). However, the immune landscape of breast cancer brain metastasis (BCBM) remains poorly defined. MATERIALS AND METHODS: The tumour-infiltrating lymphocytes (TILs) and the messenger RNA (mRNA) levels of 770 immune-related genes (NanoString™, nCounter™ Immuno-oncology IO360) were assessed in primary BCs and BCBMs. The prognostic role of ARG2 transcripts and protein expression in primary BCs and its association with outcome was determined. RESULTS: There was a significant reduction of TILs in the BCBMs in comparison to primary BCs. 11.5% of BCs presented a high immune infiltrate (hot), 46.2% were altered (immunosuppressed/excluded) and 34.6% were cold (no/low immune infiltrate). 3.8% of BCBMs were hot, 23.1% altered and 73.1% cold. One hundred and twelve immune-related genes including PD-L1 and CTLA4 were decreased in BCBM compared to the primary BCs (false discovery rate <0.01, log2 fold-change >1.5). These genes are involved in matrix remodelling and metastasis, cytokine-chemokine signalling, lymphoid compartment, antigen presentation and immune cell adhesion and migration. Immuno-modulators such as PD-L1 (CD274), CTLA4, TIGIT and CD276 (B7H3) were decreased in BCBMs. However, PD-L1 and CTLA4 expression was significantly higher in TN BCBMs (P = 0.01), with CTLA4 expression also high in human epidermal growth factor receptor 2-positive (P < 0.01) compared to estrogen receptor-positive BCBMs. ARG2 was one of four genes up-regulated in BCBMs. High ARG2 mRNA expression in primary BCs was associated with worse distant metastasis-free survival (P = 0.038), while ARG2 protein expression was associated with worse breast-brain metastasis-free (P = 0.027) and overall survival (P = 0.019). High transcript levels of ARG2 correlated to low levels of cytotoxic and T cells in both BC and BCBM (P < 0.01). CONCLUSION: This study highlights the immunological differences between primary BCs and BCBMs and the potential importance of ARG2 expression in T-cell depletion and clinical outcome.

Aberrant expression of metastasis-inducing proteins in ectopic and matched eutopic endometrium of women with endometriosis: implications for the pathogenesis of endometriosis.

BACKGROUND: Endometriosis is a metastatic disease without obvious tumorigenesis. Expression of S100P, S100A4, osteopontin (OPN) or anterior gradient homologue 2 (AGR2) proteins can induce metastasis but fail to induce tumorigenesis per se. We now explore whether this group of metastasis-inducing proteins (MIPs) are associated with the pathogenesis of endometriosis. METHODS: Eutopic endometrial biopsies were taken from 73 women (35 fertile women without endometriosis and 38 women with surgically diagnosed endometriosis). Ectopic endometriotic lesions were collected from eight of the women with endometriosis. The expression of MIPs at the cellular level was evaluated by immunohistochemistry and the presence of these proteins in the endometrial tissues was verified by western blotting and their gene expression was confirmed by RT-PCR. RESULTS: All four MIPs were immunolocated in the endometrium of control women and S100P, AGR2 and OPN showed a cyclical variation. Proliferative phase eutopic endometrium of both groups showed a similar staining pattern for all MIPs, whereas secretory phase endometrium showed a differential expression between controls and cases. The secretory phase endometrial immunostaining of controls showed weak stromal and perivascular AGR2, and decreased stromal and glandular S100P. In contrast, immunostaining for all MIPs was increased in the late secretory endometrial samples of women with endometriosis and intense immunostaining was seen for S100A4 in the stroma (P< 0.05) and for S100P (P< 0.001) and AGR2 (P< 0.0001) in both glands and stroma (P< 0.001). All active peritoneal endometriotic lesions showed strong immunostaining for each of the MIPs studied. CONCLUSIONS: We propose that these MIPs enhance endometrial cell invasiveness and contribute to the establishment of ectopic endometriotic deposits after retrograde menstruation.

RAN GTPase is an effector of the invasive/metastatic phenotype induced by osteopontin.

Osteopontin (OPN) is a phosphorylated glycoprotein that binds to alpha v-containing integrins and is important in malignant transformation and cancer. Previously, we have utilized suppressive subtractive hybridization between mRNAs isolated from the Rama 37 (R37) rat mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN to identify RAN GTPase (RAN) as the most overexpressed gene, in addition to that of OPN. Here we show that transfection of noninvasive R37 cells with an expression vector for RAN resulted in increased anchorage-independent growth, cell attachment and invasion through Matrigel in vitro, and metastasis in syngeneic rats. This induction of a malignant phenotype was induced independently of the expression of OPN, and was reversed by specifically reducing the expression of RAN using small-interfering RNAs. By using a combination of mutant protein and inhibitors, it was found that RAN signal transduction occurred through the c-Met receptor and PI3 kinase. This study therefore identifies RAN as a novel effector of OPN-mediated malignant transformation and some of its downstream signaling events in a mammary epithelial model of cancer invasion/metastasis.

Increased expression of anterior gradient-2 is significantly associated with poor survival of prostate cancer patients.

Anterior gradient-2 (AGR2) expression was examined in a series of prostate cell lines and in an archival set of prostate tissues. The relative levels of AGR2 expression in the malignant cell lines PC-3 and PC-3M were, respectively, 5.3+/-0.1 and 3.8+/-0.2 times that detected in the benign cell line PNT-2. Immunohistochemical staining in 106 cases showed that amongst seven normal cases, one (14.3%) was unstained, five (71.4%) stained weakly positive and one (14.3%) stained moderately positive. Amongst 34 benign prostate hyperplastic (BPH) cases, 12 (35.3%) were unstained, 18 (52.9%) stained weakly positive and four (11.8%) stained moderately positive. Amongst 65 carcinomas, three (4.6%) were unstained, 14 (21.5%) stained weakly positive, 19 (29.2%) stained moderately positive and 29 (44.9%) stained strongly positive. AGR2 expression in carcinomas was significantly higher than that in BPH (chi(2)-test, P<0.001). Kaplan-Meier survival analysis showed that increased AGR2 expression was significantly (log rank test, P=0.007) associated with reduced patient-survival time. Increased joint Gleason score (GS) was significantly (log rank test, P=0.001) associated with poor patient survival. However, neither prostate specific antigen (PSA) level, nor androgen receptor (AR) index, was significantly associated with patient-survival time. Increased AGR2 expression was significantly correlated with high GS (two-sided Fisher's exact test, P<0.001) and PSA levels (Mann-Whitney U-test, P=0.047), but not significantly related to the level of AR (Mann-Whitney U-test, P=0.286). These results suggest that increased AGR2 expression is a valuable prognostic factor to predict the clinical outcome of the prostate cancer patients.

Significance of the metastasis-inducing protein AGR2 for outcome in hormonally treated breast cancer patients.

The anterior gradient protein-2 (AGR2) is inducible by oestrogen and itself can induce metastasis in a rat model for breast cancer. Here, a rabbit antibody to recombinant human AGR2 was used to assess its prognostic significance in a retrospective cohort of 351 breast cancer patients treated by adjuvant hormonal therapy. The antibody stains 66% of breast carcinomas to varying degrees. The percentage of positive carcinoma cells in tumours directly correlates with the level of AGR2 mRNA (Spearman's rank correlation, P = 0.0007) and protein (linear regression analysis r2 = 0.95, P = 0.0002). There is a significant association of staining of carcinomas for AGR2 with oestrogen receptor alpha (ERalpha) staining and with low histological grade (both Fisher's Exact test P<0.0001). In the ERalpha-positive cases, but not the ERalpha-negative cases, when subdivided into the separate staining classes for AGR2, there is a significantly progressive decrease in patient survival with increased staining (log rank test, P = 0.006). The significant association of staining for AGR2 with patient death over a 10-year period (log rank test P = 0.007, hazard ratio = 3) only becomes significant at 6 years of follow-up. This may be due to the cessation of adjuvant hormonal therapy at an earlier time, resulting in adverse re-expression of the metastasis-inducing protein AGR2.

Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines.

A metastatic phenotype can be induced in benign rat mammary cells (Rama 37 cells) by transfecting them with metastasis-inducing DNAs (Met-DNAs). Stable transfection of Met-DNAs increases the level of the metastasis-associated protein, osteopontin. Randomly picked clonal cell lines have been established from the pool of Rama 37 cells transfected with one metastasis-inducing DNA, C9-Met-DNA. In these cell lines, moderate correlation is observed between the copy number of C9-Met-DNA and their metastatic potential (linear regression coefficient, R(2)=0.48). A very close correlation is observed between the cell lines' metastatic potential in vivo and the osteopontin mRNA levels in vitro (R(2)=0.74), but not with another metastasis-associated protein in this system, S100A4 (R(2)=0.21). A close correlation is also observed between osteopontin mRNA levels and the adhesive potential (R(2)=0.91) of the cells, but not with their growth rate in vitro (R(2)=0.03). These observations support the previous suggestion that osteopontin is the direct effector of C9-Met-DNA and that the presence of C9-Met-DNA is necessary, if not sufficient, for the induction of metastasis in vivo in this system. Additionally, these results suggest that Rama 37 cells with increased osteopontin mRNA levels become metastatic not through an increased growth rate, but through an increase in cellular adhesiveness.

S100A4 regulates cell motility and invasion in an in vitro model for breast cancer metastasis.

Elevated levels of the calcium-binding protein S100A4 are associated with poor patient survival in breast cancer patients and induce metastasis in rodent models. To investigate the effects of S100A4 on different components of the metastatic process, epithelial cells lines have been isolated from nonmalignant tumours in neu transgenic mice and from malignant tumours in neu/S100A4 double transgenic mice. Additional cell lines expressing both Neu and S100A4 have also been derived by transfection of rat S100A4 cDNA into tumour cell lines cloned from neu single transgenic mice. Using these cells in transfilter migration assays, it has been shown that increases in either motility or invasive properties correlate with each other and with the level of S100A4 protein. Injection of three of the cell lines separately into the mammary fat pads of nude mice showed that elevated levels of S100A4 correlated with the degree of metastasis to the lungs. In contrast, changes in cell proliferation and cell-substrate adhesion did not correlate with S100A4 levels. Neither motility nor invasiveness correlated with proteolytic degradation of gelatin as measured by zymography. Thus, the results suggest that the main effect of increases in S100A4 levels in metastasis is to generate increased cell motility and invasion and that this latter change is not dependent upon an increased ability to degrade the intercellular matrix.

Identification of mRNAs differentially-expressed between benign and malignant breast tumour cells.

Two suppression subtracted cDNA libraries have been constructed, one containing cDNAs to mRNAs present at a higher level in a benign human breast tumour-derived cell line relative to the malignant mammary cell line, MCF-7, and the other containing cDNAs present at a higher level in the MCF-7 cells relative to the benign cells. Randomly-picked cloned DNAs have been sequenced yielding 29 and 128 different cDNAs from the benign and malignant libraries, respectively. Using reverse Northern hybridisation, 76% and 83% of the cDNAs were differentially expressed by greater than two-fold, whilst 14% and 11% of cDNAs in the respective libraries were differentially expressed by more than 15-fold. Amongst these were oestrogen-responsive cDNAs and expressed sequence tags. One such oestrogen-responsive expressed sequence tag, M41, is transcribed from a gene located on chromosome 21q22.3, within an intron of a larger gene. The M41 gene contains oestrogen response elements, one of which is associated with alu repeats. M41 mRNA is expressed at a statistically significantly higher level in human breast cancer specimens than in normal human breast and benign lesions. In carcinomas, its up-regulation is associated with the development of the malignant cell.

S100A4 (p9Ka) protein in colon carcinoma and liver metastases: association with carcinoma cells and T-lymphocytes.

The presence of the EF-hand-calcium-binding protein S100A4 in the carcinoma cells of the primary tumour is associated with a shorter survival time of a group of breast cancer patients. In colon cancer, primary tumours as well as metastases to the liver can be studied. Here we show, using quantitative PCR applied to RNA from 24 normal colon, four liver tissues, 24 colon carcinoma specimens, and 24 livers containing colonic carcinoma metastases, that the level of S100A4 mRNA was significantly higher in the carcinomas compared to normal specimens (Mann-Whitney U-test, P=0.05), and in liver metastases compared to carcinoma specimens (P=0.039). The latter comparison included seven liver metastases and their matched primary carcinomas (P<0.001) from the same patient. In situ hybridization and immunocytochemistry techniques have localized S100A4 to both carcinoma cells and lymphocytes in the malignant specimens. The percentage of specimens stained for S100A4 in the epithelial cells is significantly higher for those isolated from carcinomas and metastases than from the corresponding normal tissue, and from metastases than from corresponding carcinoma (Fisher Exact text, P<0.0016, P=0.04, respectively). In most specimens, S100A4 is present in clusters of T lymphocytes and this distribution is also found in the lymphoid, uninflamed appendix.

Hepatocyte growth factor/scatter factor stimulates migration of rat mammary fibroblasts through both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways.

Hepatocyte growth factor/scatter factor (HGF/SF) is considered to be a mesenchymal-derived factor that acts via a dual system receptor, consisting of the MET receptor and proteoglycans present on adjacent epithelial cells. Surprisingly, HGS/SF stimulated the migration of rat mammary (Rama) 27 fibroblasts, although it failed to stimulate their proliferation. HGF/SF stimulated a transient activation of mitogen-activated protein kinases p44 and p42 (p42/44(MAPK)), with a maximum level of dual phosphorylation of p42/44(MAPK) occurring 10-15 min after the addition of the growth factor, which was followed by a rapid decrease to near basal levels after 20 min. Interestingly, a second phase of p42/44(MAPK) dual phosphorylation was observed at later times (3 h to 10 h). PD098059, a specific inhibitor of MEK-1, prevented the dual phosphorylation of p42/44(MAPK) and also the phosphorylation of p90(RSK) (ribosomal subunit S6 kinase), which mirrored the kinetics of p42/44(MAPK) phosphorylation. Moreover, PD098059 prevented the HGF/SF-induced migration of Rama 27 cells. HGF/SF also induced an early increase in the phosphorylation of protein kinase B/Akt. Akt phosphorylation was elevated 15 min after the addition of HGF/SF and then declined to basal levels by 30 min. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PtdIns3K), prevented the increase in Akt phosphorylation and abolished HGF/SF-induced migration of fibroblasts. PD098059 also inhibited the stimulation of Akt phosphorylation by HGF/SF and wortmannin similarly inhibited the stimulation of p42/44(MAPK) dual phosphorylation. These results suggest that HGF/SF-induced motility depends on both the transient dual phosphorylation of p42/44(MAPK) and the activation of PtdIns3K in Rama 27 fibroblasts and that these pathways are mutually dependent.

Binding to intracellular targets of the metastasis-inducing protein, S100A4 (p9Ka).

Experimentally elevated levels of S100A4 induce a metastatic phenotype in benign mammary tumour cells in vivo. In humans, the presence of S100A4 in breast cancer cells correlates strongly with reduced patient survival. Potential interacting binding partners for S100A4 have now been examined using an optical biosensor. There was significant interaction of S100A4 with non-muscle myosin and p53, but not with actin, tropomyosin or tubulin. The results suggest that myosin and p53 are likely to be intracellular targets of S100A4. S100A4 had a greater affinity for wild-type or mutant arg-175-his p53 than for non-muscle myosin. The results suggest that S100A4 might induce metastasis by influencing the function of p53 as well as through its interaction with myosin and that any mechanism is independent of the mutational status of p53.

Metastasis-inducing dna regulates the expression of the osteopontin gene by binding the transcription factor Tcf-4.

Small 1,000-bp fragments of genomic DNA obtained from human malignant breast cancer cell lines when transfected into a benign rat mammary cell line enhance transcription of the osteopontin gene and thereby cause the cells to metastasize in syngeneic rats. To identify the molecular events underlying this process, transient cotransfections of an osteopontin promoter-reporter construct and fragments of one metastasis-inducing DNA (Met-DNA) have identified the active components in the Met-DNA as the binding sites for the T-cell factor (Tcf) family of transcription factors. Incubation of cell extracts with active DNA fragments containing the sequence CAAAG caused retardation of their mobilities on polyacrylamide gels, and Western blotting identified Tcf-4, beta-catenin, and E-cadherin in the relevant DNA complexes in vitro. Transfection of an expression vector for Tcf-4 inhibited the stimulated activity of the osteopontin promoter-reporter construct caused by transiently transfected active fragments of Met-DNA or permanently transfected Met-DNA. This stimulated activity of the osteopontin promoter-reporter construct is accompanied by an increase in endogenous osteopontin mRNA but not in fos or actin mRNAs in the transfected cells. Permanent transfection of the benign rat mammary cell line with a 20-bp fragment from the Met-DNA containing the Tcf recognition sequence CAAAG caused an enhanced permanent production of endogenous osteopontin protein in vitro and induced the cells to metastasize in syngeneic rats in vivo. The corresponding fragment without the CAAAG sequence was without either effect. Therefore, the regulatory effect of the C9-Met-DNA is exerted, at least in part, by a CAAAG sequence that can sequester the endogenous inhibitory Tcf-4 and thereby promote transcription of osteopontin, the direct effector of metastasis in this system.

Human cutaneous fatty acid-binding protein induces metastasis by up-regulating the expression of vascular endothelial growth factor gene in rat Rama 37 model cells.

Human cutaneous fatty acid-binding protein (C-FABP) gene is capable of inducing the metastatic phenotype when overexpressed in nonmetastatic rat Rama 37 cells. However, the mechanism of how it induces metastasis is not clear. Northern and slot blot analyses revealed that expression of the endogenous vascular endothelial growth factor (VEGF) gene was increased by 3.8-5.2-fold in the C-FABP-transfected cells (pSV-CFABP-R37) and in their metastatic sublines (e.g., Met-1) when compared with that in the nonmetastatic control transfectant pSV-R37 cells generated by transfection of only plasmid DNA. Higher levels of VEGF immunoreactive protein were also secreted from the malignant C-FABP-expressing cells. Reverse transcription-PCR detected two VEGF transcript isoforms, VEGF(164) and VEGF(188), in both the nonmetastatic control transfectant pSV-R37 cells and the malignant metastatic Met-1 cells. Chick chorioallantoic membrane assays showed that the conditioned medium of the control pSV-R37 cells possessed only very weak angiogenic activity, whereas conditioned media from the metastatic C-FABP transfectants and their sublines were strongly angiogenic and could be inhibited by antibodies to VEGF. Transfection of VEGF(164) cDNA in an expression vector into nonmetastatic Rama 37 cells produced a cell clone (R37-VEGF-2) that expressed high levels of VEGF. Inoculation of R37-VEGF-2 cells into syngeneic Wistar Furth rats produced metastases in a significant number (Fisher's exact test, P < 0.01) of animals (18 of 31 animals), whereas the control, vector alone-transfected R37-PSV cells produced no metastases (0 of 30 animals). Immunocytochemical methods demonstrated a strong positive staining for VEGF and an increased microvessel density in the primary tumors produced from PSV-VEGF-2 cells in comparison with tumors produced from control transfectants. Immunocytochemical staining for factor VIII detected a 3.5-fold increase in microvessel density of the primary tumors produced by PSV-VEGF-2 cells when compared with that of the primary tumors developed from the control pSV-R37 cells. Therefore, we suggest that overexpression of the C-FABP gene in the original transfectants induces metastasis through up-regulation of expression of the VEGF gene in this rat Rama 37 model system, and thus VEGF may play a crucial role in this particular metastatic cascade.

Preliminary X-ray crystallographic analysis of a Ca2+-binding protein human S100A1.

S100A1, a Ca(2+)-binding protein from the S100 protein family, has been crystallized by the vapour-diffusion method using polyethylene glycol 4000 as the precipitant at pH 8.5. The crystal belongs to space group P6(3). The unit-cell parameters are a = b = 57.3, c = 104.7 A. There appear to be two S100A1 molecules in the asymmetric unit. The crystals were stable during exposure to X-rays and diffract to 2.6 A resolution in-house.

Regulatory region of metastasis-inducing DNA is the binding site for T cell factor-4.

Small 1000 bp fragments of DNA derived from human malignant breast cancer cells have been isolated which, when transfected into a benign rat mammary cell line induce the production of osteopontin and thereby endow those cells with the capability to metastasize in syngeneic rats. Using transient transfections of an osteopontin promoter-reporter construct, we have now identified the active moiety in the metastasis-inducing DNA as the binding site for the T cell factor (Tcf) family of transcription factors and located Tcf-4, beta-catenin and E-cadherin in the relevant DNA complex in vitro. The regulatory effects of the metastasis-inducing DNAs are therefore exerted, at least in part, by a CAAAG sequence which can sequester Tcf-4, thereby promoting transcription of the direct effector for metastasis in this system, osteopontin.

Sialomucin complex (rat Muc4) transmembrane subunit binds the differentiation marker peanut lectin in the normal rat mammary gland.

Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein composed of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. SMC/Muc4 is highly expressed on the surface of 13762 rat mammary adenocarcinoma cells at approximately 100 times the level found in the lactating mammary gland. Immunocytochemical staining of SMC/Muc4 in the developing rat mammary gland is localized to the apical membrane of the ductal epithelium. This staining pattern is similar to that for peanut lectin, a differentiation marker, which binds to cells expressing the disaccharide Thomsen-Friedenreich or TF antigen. Blotting of glycoproteins expressing the TF antigen from mammary tissues with peanut lectin detects a protein matching the migration of ASGP-2. Analysis of immunoprecipitated SMC/Muc4 by peanut lectin blotting shows that the TF antigen is abundantly present on the ASGP-2 subunit, hence the similarity of staining pattern with SMC/Muc4 antisera and peroxidase-conjugated lectin in mammary tissues. The TF antigen is also present on ASGP-2 of SMC/Muc4 produced by confluent cultures of Rama 37 rat mammary epithelial stem cells after their induction to an alveolar-like phenotype with prolactin. These results indicate that the TF antigen is present on the ASGP-2 transmembrane subunit of SMC/Muc4 from phenotypically normal tissues and cells, in contrast to malignant cells whose peanut lectin-binding disaccharide is located on ASGP-1.

Expression of calcium-binding protein S100A2 in breast lesions.

A suppression subtraction cDNA library representing mRNAs expressed at a higher level in a benign breast tumour-derived cell line relative to the malignant MCF-7A cell line contained cDNAs corresponding to mRNAs for plasminogen activator inhibitor I, annexin VIII and the EF-hand protein S100A2. S100A2 protein has previously been shown to be expressed in normal human breast epithelium, but not in human breast carcinoma cell lines. Using a PCR-based assay and in situ hybridization on histological sections of human breast specimens, the mRNA for S100A2 was shown to be present in all benign breast lesions examined as well as in normal epithelium. S100A2 mRNA was detectable in 37% of specimens of carcinoma in situ, but in less than 15% of carcinoma specimens. The results suggest that the loss of S100A2 is associated with the development of malignant cells and is not associated with early tumour development.

Variant estrogen receptor alpha mRNAs in human breast cancer specimens.

A panel of human breast cancer specimens was examined for single base change mutations by DNA sequencing and for larger deletions using a PCR-based assay. In the cancer specimens examined, no sequencing variants were detected other than a previously characterized polymorphism. Although most of the specimens contained estrogen receptor (ER) variants at a low level, 2 of 118 specimens exhibited variants which, after amplification, constituted most of the amplified ER cDNA. One specimen contained a single variant form, and there was little evidence of the wild-type ER mRNA by PCR, Northern blotting or immunocytochemistry. The second specimen, despite the presence of a normal-sized mRNA by Northern blotting and normal immunocytochemical staining for ER, contained at least 5 different variant forms as well as the wild-type ER. All but 1 of the variant forms were processing variants, and 3 of these processing variants have not been described before. One variant, although lacking exons 2-4, has break points in exons 1 and 5 that do not correspond to intron-exon boundaries. This variant might reflect more widespread damage to the genome in this breast cancer specimen. The low level of occurrence of variants suggests that ER variant forms, at least in the coding region, do not contribute generally to the progression of breast cancer.

Fibroblast growth factor-2 stimulation of p42/44MAPK phosphorylation and IkappaB degradation is regulated by heparan sulfate/heparin in rat mammary fibroblasts.

Fibroblast growth factor-2 (FGF-2) interacts with a dual receptor system consisting of tyrosine kinase receptors and heparan sulfate proteoglycans (HSPGs). In rat mammary fibroblasts, FGF-2 stimulated DNA synthesis and induced a sustained phosphorylation of p42/44(MAPK) and of its downstream target, p90(RSK). Moreover, FGF-2 also stimulated the transient degradation of IkappaBalpha and IkappaBbeta. PD098059, a specific inhibitor of p42/44(MAPK) phosphorylation, inhibited FGF-2-stimulated DNA synthesis, phosphorylation of p42/44(MAPK) and p90(RSK), and degradation of IkappaBbeta. In contrast, in chlorate-treated and hence sulfated glycosaminoglycan-deficient cells, FGF-2 was unable to stimulate DNA synthesis. However, FGF-2 was able to trigger a transient phosphorylation of both p42/44(MAPK) and p90(RSK), which peaked at 15 min and returned to control levels at 30 min. In these sulfated glycosaminoglycan-deficient cells, no degradation of IkappaBalpha and IkappaBbeta was observed after FGF-2 addition. However, in chlorate-treated cells, the addition of heparin or purified HSPGs simultaneously with FGF-2 restored DNA synthesis, the sustained phosphorylation of p42/44(MAPK) and p90(RSK), and the degradation of IkappaBalpha and IkappaBbeta. These results suggest that the HSPG receptor for FGF-2 not only influences the outcome of FGF-2 signaling, e.g. cell proliferation, but importantly regulates the immediate-early signals generated by this growth factor.