Matriptase activation and shedding through PDGF-D-mediated extracellular acidosis

Abstract

Activation of β-platelet-derived growth factor receptor (β-PDGFR) is associated with prostate cancer (PCa) progression and recurrence after prostatectomy. Analysis of the β-PDGFR ligands in PCa revealed association between PDGF-D expression and Gleason score as well as tumor stage. During the course of studying the functional consequences of PDGF ligand-specific β-PDGFR signaling in PCa, we discovered a novel function of PDGF-D for activation/shedding of the serine protease matriptase leading to cell invasion, migration, and tumorigenesis. The present study showed that PDGF-D, not PDGF-B, induces extracellular acidification, which correlates with increased matriptase activation. A cDNA microarray analysis revealed that PDGF-D/β-PDGFR signaling upregulates expression of the acidosis regulator carbonic anhydrase IX (CAIX), a classic target of the transcriptional factor hypoxia-inducible factor-1α (HIF-1α). Cellular fractionation displayed a strong HIF-1α nuclear localization in PDGF-D-expressing cells. Treatment of vector control or PDGF-B-expressing cells with the HIF-1α activator CoCl2 led to increased CAIX expression accompanied by extracellular acidosis and matriptase activation. Furthermore, the analysis of the CAFTD cell lines, variants of the BPH-1 transformation model, showed that increased PDGF-D expression is associated with enhanced HIF-1α activity, CAIX induction, cellular acidosis, and matriptase shedding. Importantly, shRNA-mediated knockdown of CAIX expression effectively reversed extracellular acidosis and matriptase activation in PDGF-D-transfected BPH-1 cells and in CAFTD variants that express endogenous PDGF-D at a high level. Taken together, these novel findings reveal a new paradigm in matriptase activation involving PDGF-D-specific signal transduction leading to extracellular acidosis.

Keywords: CAIX; HIF-1α; PDGF-D; acidosis; matriptase.

Fig. 1.

Platelet-derived growth factor-DPDGF-D-specific signaling induces extracellular acidosis in prostate epithelial cells. A: immunoblot analysis of matriptase and with hepatocyte growth factor activator inhibitor-1 (HAI-1) in conditioned media and total cell lysates from serum-starved vector control (Hygro), PDGF-B BPH-1, or PDGF-D BPH-1 cells. B: changes in extracellular pH of vector control (Hygro), PDGF-B BPH-1, or PDGF-D BPH-1 cells are plotted. Lines represent the mean ± SD. *P < 0.05. C: change in extracellular pH of vector control (Hygro), PDGF-B BPH-1, or PDGF-D BPH-1 cells was normalized to cell number. Lines represent the mean ± SD. *P < 0.05, comparing vector control (Hygro) and PDGF-B cells; **P < 0.05, comparing vector (Hygro) and PDGF-D cells. aMat, active matriptase; tMat, total matriptase. Ponceau S was used to evaluate equal gel loading of conditioned media.

Fig. 2.

Matriptase activation/shedding is induced by extracellular acidosis in prostate epithelial cells. A: immunoblot of active and total matriptase in conditioned media from vector control (Hygro) or PDGF-B BPH-1 cells upon treatment with pH 7.4 or pH 6.0 phosphate buffer for indicated time period. Intracellular pH of vector control (Hygro), PDGF-B BPH-1, and PDGF-D BPH-1 cells was monitored through membrane zeta potential (B) and BCECF fluorescent (C) measurements. Lines represent the mean ± SD. *P < 0.05. Ponceau S was used to evaluate equal gel loading of conditioned media.

Fig. 3.

Enhanced PDGF-D expression in the BPH-1 transformation model correlates with matriptase shedding. A: immunoblot analysis of PDGF-D and PDGF-B in conditioned media or cell lysates from serum-starved parental BPH-1, BPH-1 CAFTD-3, and BPH-1 CAFTD-4 variants. B: BPH-1 CAFTD-4 conditioned media (CM) were incubated with 5 ng recombinant matriptase (rMat) for 2 or 6 h. Conditioned media alone were used as a digest control. PDGF-D processing was assessed using immunoblot analysis. Ponceau S was used as a loading control. C: NIH3T3 cells were treated with digested or undigested conditioned media from parental or BPH-1 CAFTD-4 cells and β-PDGFR activation assessed. rPDGF-B and serum free media (SFM) alone were used as positive and negative controls, respectively. Numbers under phospho-PDGFRβ and Akt are relative densitometric values obtained using National Institutes of Health ImageJ. D: matriptase expression profile in the BPH-1 transformation model. Ponceau S was used to evaluate equal gel loading of conditioned media.

Fig. 4.

Extracellular acidosis and increased cell invasion are evident in the BPH-1 transformation model cells expressing high PDGF-D. Changes in extracellular pH (A) were monitored in BPH-1 parental and BPH-1 variant cells then normalized to cell number (B). C: Matrigel invasion was assessed in BPH-1 parental and BPH-1 variant cells. Values represent the mean ± SD. HPF, high-power field. *P < 0.05, between BPH-1 parental and BPH-1 CAFTD-3; **P < 0.05, between BPH-1 parental and BPH-1 CAFTD-4.

Fig. 5.

Carbonic anhydrase IX (CAIX) expression is enhanced in response to PDGF-D upregulation. A: list of pH regulating genes from a cDNA microarray analysis of vector control (Hygro), PDGF-B BPH-1, and PDGF-D BPH-1 cells. Qualitative and quantitative RT-PCR as well as immunoblot analysis of carbonic anhydrase (CA) family members in vector control (Hygro), PDGF-B and PDGF-D BPH-1 (B and C), and BPH-1 CAFTD variant (D and E) cells. *P < 0.05.

Fig. 6.

CAIX downregulation abrogates PDGF-D-mediated extracellular acidosis. PDGF-D BPH-1 (A) or BPH-1 CAFTD-4 (D) cells were transfected with scrambled (shScrm) or two separate CAIX shRNA and its knockdown was monitored via immunoblotting. Changes in extracellular pH were monitored then normalized to cell number in control and shRNA-mediated CAIX knockdown PDGF-D BPH-1 (B and C) or BPH-1 CAFTD-4 (E and F). Bars represent the mean ± SD. *P < 0.05, between shScrm and shCAIX-2; **P < 0.05, between shScrm and shCAIX-3.

Fig. 7.

Matriptase shedding and cell invasion is attenuated in response to CAIX knockdown. Immunoblot analysis of matriptase and HAI-1 using conditioned media of PDGF-D BPH-1 (A) and BPH-1 CAFTD-4 (B) cells without or with CAIX knockdown (shScrm and shCAIX, respectively). Ponceau S was used to evaluate equal gel loading. Matrigel invasion was monitored in control and shRNA-mediated CAIX knockdown PDGF-D BPH-1 (C) and BPH-1 CAFTD-4 (D) cells. Bars represent the mean ± SD. *P < 0.05, between shScrm and shCAIX-2; **P < 0.05, between shScrm and shCAIX-3.

Fig. 8.

PDGF-D signaling upregulates nuclear hypoxia-inducible factor-1α (HIF-1α). A: RT-PCR analysis of HIF-1α expression in vector control (Hygro), PDGF-B BPH-1, or PDGF-D BPH-1 cells. Immunoblot analyses of HIF-1α in nuclear fraction (Nuc) and cytoplasmic fraction (Cyt) of vector control (Hygro), PDGF-B BPH-1, and PDGF-D BPH-1 cells (B) or in the parental BPH-1 and CAFTD-4 variant (C). Histone H1 and PTEN were used as loading controls for the nuclear and cytoplasmic fractions, respectively.

Fig. 9.

HIF-1α activation supports extracellular acidosis. A: immunoblot analysis of HIF-1α (arrow) in the nuclear and cytoplasmic fractions from vector control (Hygro) or PDGF-B BPH-1 cells with vehicle only or 100 μM CoCl₂ treatment. Histone H1 and PTEN were used as loading controls for the nuclear and cytoplasmic fractions, respectively. Upon treatment of vector (Hygro) or PDGF-B BPH-1 cells with CoCl₂, CA mRNA expression (RT-PCR) (B), changes in extracellular pH (C–F), and matriptase activation (G) were monitored. Values represent the mean ± SD. *P < 0.05. Ponceau S was used to evaluate equal gel loading.

Fig. 10.

A working model of PDGF-D-mediated matriptase activation. Matriptase activates full length PDGF-D (FL-PDGF-D) in a biphasic manner first yielding a PDGF-D hemidimer (HD-PDGF-D) then growth factor only PDGF-D (GD-PDGF-D). PDGF-D/β-PDGFR signaling mediates HIF-1α nuclear translocation and transcription of CAIX leading to extracellular acidosis (H⁺) supporting matriptase activation and enhancing further PDGF-D processing and driving a proinvasive program. L-Mat, latent matriptase.

Fig. 11.

Elucidation of PDGF-D-mediated HIF-1α nuclear localization. Hygro vector control, PDGF-B BPH-1, and PDGF-D BPH-1 cells were treated with 2 μg/ml cycloheximide (Cyclo) for 2 h to inhibit protein synthesis. Cycloheximide was then removed and HIF-1α localization was monitored in the presence or the absence of 50 μM MG132 (MG) at 0.5 and 4 h. Histone H1 and PTEN were used as loading controls for the nuclear (A) and cytoplasmic (B) fractions, respectively.