Platelet-derived growth factor-C (PDGF-C) activation by serine proteases: implications for breast cancer progression - PubMed (original) (raw)

Platelet-derived growth factor-C (PDGF-C) activation by serine proteases: implications for breast cancer progression

Newton J Hurst Jr et al. Biochem J. 2012.

Abstract

The PDGF (platelet-derived growth factor) family members are potent mitogens for cells of mesenchymal origin and serve as important regulators of cell migration, survival, apoptosis and transformation. Tumour-derived PDGF ligands are thought to function in both autocrine and paracrine manners, activating receptors on tumour and surrounding stromal cells. PDGF-C and -D are secreted as latent dimers, unlike PDGF-A and -B. Cleavage of the CUB domain from the PDGF-C and -D dimers is required for their biological activity. At present, little is known about the proteolytic processing of PDGF-C, the rate-limiting step in the regulation of PDGF-C activity. In the present study we show that the breast carcinoma cell line MCF7, engineered to overexpress PDGF-C, produces proteases capable of cleaving PDGF-C to its active form. Increased PDGF-C expression enhances cell proliferation, anchorage-independent cell growth and tumour cell motility by autocrine signalling. In addition, MCF7-produced PDGF-C induces fibroblast cell migration in a paracrine manner. Interestingly, PDGF-C enhances tumour cell invasion in the presence of fibroblasts, suggesting a role for tumour-derived PDGF-C in tumour-stromal interactions. In the present study, we identify tPA (tissue plasminogen activator) and matriptase as major proteases for processing of PDGF-C in MCF7 cells. In in vitro studies, we also show that uPA (urokinase-type plasminogen activator) is able to process PDGF-C. Furthermore, by site-directed mutagenesis, we identify the cleavage site for these proteases in PDGF-C. Lastly, we provide evidence suggesting a two-step proteolytic processing of PDGF-C involving creation of a hemidimer, followed by GFD-D (growth factor domain dimer) generation.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1. Expression and processing of platelet-derived growth factor-C (PDGF-C) in MCF7

A. PDGF-C levels in MCF7-PDGF-C (Lane 1) and -Neo (Lane 2) were examined by RT-PCR. B. Serum-free (SF) conditioned media (CM) collected from MCF7-PDGF C or -Neo cells were analyzed by immunoblot analysis using anti-PDGF-C growth factor domain Ab in reducing (left panel) and non-reducing (right panel) conditions. FL-M, full-length monomer; GFD-M, growth factor domain monomer; FL-D, full-length dimer; GFD-D, growth factor domain dimer. C. Serum-starved NIH3T3 fibroblasts were stimulated for 10 min with CM collected from MCF7-PDGF-C (Lane 1) or MCF7-Neo (Lane 2) cells, SF media with 20ng/ml PDGF-AA (Lane 3, positive control), or SF media (Lane 4, negative control). Cell lysates were immunoprecipitated using anti-PDGF-C Ab and activated α-PDGFR was detected by immunoblot using a pTyr Ab.

Figure 2

Figure 2. In vitro transformative properties of PDGF-C in MCF7 cells

A (paracrine signaling). A scratch migration assay of NIH3T3 was performed in the presence of conditioned media collected from MCF7-neo or –PDGF-C cells. Using NIH ImageJ, closure of the gap was quantified as a percentage of cleared area remaining at time 0, 8, and 16 from three independent experiments (top panel). Representative 40X images of time 0 and 16 hours are displayed (bottom panel). B and C (autocrine signaling). Anchorage-independent growth and proliferation of MCF7-neo or –PDGF-C cells were assessed by a soft agar colony formation assay (B) and WST-1 cell proliferation assay (C), respectively. Positive colonies were quantified from three separate experiments using Optronix GelCount (Oxford, England) in panel B and cell proliferation was quantified from three independent WST-1 assays in panel C. D (autocrine and double paracrine signaling). Matrigel invasion assay of MCF7-neo or –PDGF-C cells through a modified Boyden chamber was performed in the absence or presence of NIH3T3 fibroblasts in the bottom chamber. Quantitation is averaged results of three separate experiments. *p<0.05.

Figure 3

Figure 3. PDGF-C is processed by serine proteases, specifically tPA and uPA

A. MCF7-PDGF-C cells were incubated in SF media with the various class specific inhibitors for 48h; the collected CM was resolved under reducing SDS-PAGE and immunoblotted with an anti-PDGF-C GFD Ab. B. Left panel, MCF7-PDGF-C cells were incubated with SF media without or with PAI-1. Right panel, MCF7-PDGF-C (Lanes 1 and 3) or MCF7-neo (Lanes 2 and 4) cells were incubated with SF media containing tPA or uPA specific inhibitors for 48h; the collected CM was resolved under reducing SDS-PAGE and immunoblotted with an anti-PDGF-C GFD Ab. C. MCF7-PDGF-C (Lane 3) and MCF7-Neo (Lane 4) CM was run in a plasminogen-dependent zymogram with r-tPA (Lane 1) and r-uPA (Lane 2) serving as positive activity controls. D. rPDGF-C was generated by co-infecting/transfecting CV-1 cells with vaccinia virus and the pTF7-PDGF-C: HIS construct. After 48h of serum-starvation the CM was collected. This CM was concentrated using Ni-NTA agarose beads overnight. After washing of the beads, rPDGF-C was eluted with 10mM EDTA then rPDGF-C was incubated with tPA or uPA overnight. Finally, the resultant products were resolved by SDS-PAGE and immunoblotted with an anti-PDGF-C GFD Ab. E. rPDGF-C was incubated with tPA, uPA, streptokinase, and streptokinase+glu-plasminogen overnight, and the resultant products were resolved by SDS-PAGE and immunoblotted with an anti-PDGF-C GFD Ab.

Figure 4

Figure 4. Mutational analysis identifies tPA and uPA cleavage sites

A. Comparison of sequence alignments between human PDGF-C, murine PDGF-C, and human PDGF-D. Mutagenesis sites are marked with asterisks and the tPA/uPA substrate specificity in the P1–P4 residues are shown. B. Western blot analysis of PDGF-C wild-type (WT), PDGF-C K225A, PDGF-C R231A/234A, and PDGF-C K225A/R231A/R234A mutants under reducing (left panel) and non-reducing (right panel) conditions when incubated with 100nM of tPA 4h at 37°C in the presence of fibrinogen fragments. C. Western blot analysis of PDGF-C WT, PDGF-C K225A, PDGF-C R231A/R234A, and PDGF-C K225A/R231A/R234A under reducing (left panel) and non-reducing (right panel) conditions when incubated with 100nM of uPA overnight at 37°C.

Figure 5

Figure 5. Matriptase is capable of processing PDGF-C

A. Western blot analysis of MCF7-PDGF-C and -Neo CM and lysates for matriptase expression. Blot was probed with anti-matriptase Ab (kind gift of Dr. C-Y Lin, University of Maryland) under the non-reducing condition. B. rPDGF-C WT incubated with varying concentrations of matriptase 2h at 37°C and then resolved on SDS-PAGE under reducing conditions C. rPDGF-C WT and mutants were incubated with 1nM matriptase for 2h at 37°C and then resolved on SDS-PAGE under reducing conditions (top panel) or non-reducing conditions (bottom panel). D. rPDGF-C WT or R231A/R234A were incubated 2h at 37°C with 1nM matriptase before being suspended in SF DMEM/F12 media and then added to NIH3T3 cells for 10 min. Subsequently, the lysates from these cells were analyzed by western blot for the presence of phospho-β-PDGFR. Total β-PDGFR was used as a loading control. MCF7-PDGF-C cells were treated with the matriptase inhibitor, HAI-1, and PDGF-C processing (E) and biological activity (F) were monitored. In the same gel, an unnecessary lane separating SFM and NT treatment was removed in panel E. A white line is drawn to demarcate this change. G. Conditioned media from (E) were utilized to assess NIH3T3 cell migration. * and ** p<0.05.

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References

    1. Deuel TF. Polypeptide growth factors: roles in normal and abnormal cell growth. Annu Rev Cell Biol. 1987;3:443–492. - PubMed
    1. Ross R, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell. 1986;46:155–169. - PubMed
    1. Rosenkranz S, Kazlauskas A. Evidence for distinct signaling properties and biological responses induced by the PDGF receptor alpha and beta subtypes. Growth Factors. 1999;16:201–216. - PubMed
    1. Anderberg C, Li H, Fredriksson L, Andrae J, Betsholtz C, Li X, Eriksson U, Pietras K. Paracrine signaling by platelet-derived growth factor-CC promotes tumor growth by recruitment of cancer-associated fibroblasts. Cancer Res. 2009;69:369–378. - PMC - PubMed
    1. Crawford Y, Kasman I, Yu L, Zhong C, Wu X, Modrusan Z, Kaminker J, Ferrara N. PDGF-C mediates the angiogenic and tumorigenic properties of fibroblasts associated with tumors refractory to anti-VEGF treatment. Cancer Cell. 2009;15:21–34. - PubMed

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