Login to Access Video or Poster Abstract: MP17-09
Sources of Funding: NIH/NIDDK 1U54 DK104310 (Ricke, PI; Macoska, Co-PI); NIH/NIDDK 1R21DK098304 (Macoska , PI)

Introduction

Factors that promote lower urinary tract voiding dysfunction (LUTD) in aging men include excessive prostatic proliferation and muscle contraction. Recent studies suggest that tissue fibrosis, e.g., peri-urethral collagen accumulation, may also contribute to LUTD. Our group recently reported that activation of the CXCL12/CXCR4 axis and downstream MEK/ERK activation, as well as canonical TGFβ/TGFβR axis activation and downstream Smad3 activation, promote prostate fibroblast to myofibroblast phenoconversion and collagen production. Based on this finding, we hypothesized that downstream MEK/ERK and Smad signaling would converge at the transcriptional level to promote the activation of genes encoding pro-fibrotic proteins. To test this, RNA sequencing analysis was performed on prostate fibroblasts treated with CXCL12 or TGFβ.

Methods

Prostate fibroblasts were treated with 4ng/ml TGFβ or 100pM CXCL12 for 12 hrs (RNA) or 24, 48 and 72 hrs (protein). Total RNA was purified and subjected to qRT-PCR or RNASeq. Sequence data was pipelined through the Bowtie/TopHat/Cufflinks/CummeRbund (Tuxedo) packages. Pathview v1.10.1 and Cytoscape were used to perform Kegg pathway and network analysis, respectively. Immunoblot analysis was performed using whole protein lysates or concentrated conditioned media.

Results

RNASeq analysis showed that prostate fibroblasts treated with CXCL12 up-regulated the transcript levels of genes that encoded members of the Cullin-RING 3 (CRL3) ubiquitin ligase family of proteins. These proteins form COPII vesicles that transport procollagen from the endoplasmic reticulum to the cell membrane and extracellular space. Western blot analysis showed that CXCL12-treated cells up-regulated CRL3 proteins and secreted higher levels of procollagen compared to vehicle and TGFβ-treated cells. Procollagen secretion was ablated upon treatment with AMD3100, a CXCR4 antagonist, demonstrating that the observed increased collagen secretion was specifically coupled to CXCL12/CXCR4 axis activation.

Conclusions

The results of these studies are consistent with our hypothesis and present a major new discovery: Activation of the CXCL12/CXCR4 axis promotes fibrosis by increasing both the expression and secretion of collagen.

Funding

NIH/NIDDK 1U54 DK104310 (Ricke, PI; Macoska, Co-PI); NIH/NIDDK 1R21DK098304 (Macoska , PI)