Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.

Heparan sulfate proteoglycan 2 (HSPG2) is an essential, highly conserved gene whose expression influences many developmental processes including the formation of the heart and brain. The gene is widely expressed throughout the musculoskeletal system including cartilage, bone marrow and skeletal muscle. The HSPG2 gene product, perlecan is a multifunctional proteoglycan that preserves the integrity of extracellular matrices, patrols tissue borders, and controls various signaling pathways affecting cellular phenotype. Given HSPG2’s expression pattern and its role in so many fundamental processes, it is not surprising that relatively few gene mutations have been identified in viable organisms. Mutations to the perlecan gene are rare, with effects ranging from a relatively mild condition to a more severe and perinatally lethal form. This review will summarize the important studies characterizing mutations and variants of HSPG2 and discuss how these genomic modifications affect expression, function and phenotype. Additionally, this review will describe the clinical findings of reported HSPG2 mutations and their observed phenotypes. Finally, the evolutionary aspects that link gene integrity to function are discussed, including key findings from both in vivo animal studies and in vitro systems. We also hope to facilitate discussion about perlecan/HSPG2 and its role in normal physiology, to explain how mutation can lead to pathology, and to point out how this information can suggest pathways for future mechanistic studies.

Perlecan/HSPG2, a large, monomeric heparan sulfate proteoglycan (HSPG), is a key component of the lacunar canalicular system (LCS) of cortical bone, where it is part of the mechanosensing pericellular matrix (PCM) surrounding the osteocytic processes and serves as a tethering element that connects the osteocyte cell body to the bone matrix. Within the pericellular space surrounding the osteocyte cell body, perlecan can experience physiological fluid flow drag force and in that capacity function as a sensor to relay external stimuli to the osteocyte cell membrane. We previously showed a reduction in perlecan secretion alters the PCM fiber composition and interferes with bone’s response to mechanical loading in vivo. To test our hypothesis that perlecan core protein can sustain tensile forces without unfolding under physiological loading conditions, atomic force microscopy (AFM) was used to capture images of perlecan monomers at nanoscale resolution and to perform single molecule force measurement (SMFMs). We found that the core protein of purified full-length human perlecan is of suitable size to span the pericellular space of the LCS, with a measured end-to-end length of 170 ± 20 nm and a diameter of 2–4 nm. Force pulling revealed a strong protein core that can withstand over 100 pN of tension well over the drag forces that are estimated to be exerted on the individual osteocyte tethers. Data fitting with an extensible worm-like chain model showed that the perlecan protein core has a mean elastic constant of 890 pN and a corresponding Young’s modulus of 71 MPa. We conclude perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS.

Interrupting the interplay between cancer cells and extracellular matrix (ECM) is a strategy to halt tumor progression and stromal invasion. Perlecan/heparan sulfate proteoglycan 2 (HSPG2) is an extracellular proteoglycan that orchestrates tumor angiogenesis, proliferation, differentiation and invasion. Metastatic prostate cancer (PCa) cells degrade perlecan-rich tissue borders to reach bone, including the basement membrane, vasculature, reactive stromal matrix and bone marrow. Domain IV-3, perlecan’s last 7 immunoglobulin repeats, mimics native proteoglycan by promoting tumoroid formation. This is reversed by matrilysin/matrix metalloproteinase-7 (MMP-7) cleavage to favor cell dispersion and tumoroid dyscohesion. Both perlecan and Domain IV-3 induced a strong focal adhesion kinase (FAK) dephosphorylation/deactivation. MMP-7 cleavage of perlecan reversed this, with FAK in dispersed tumoroids becoming phosphorylated/activated with metastatic phenotype. We demonstrated Domain IV-3 interacts with the axon guidance protein semaphorin 3A (Sema3A) on PCa cells to deactivate pro-metastatic FAK. Sema3A antibody mimicked the Domain IV-3 clustering activity. Direct binding experiments showed Domain IV-3 binds Sema3A. Knockdown of Sema3A prevented Domain IV-3-induced tumoroid formation and Sema3A was sensitive to MMP-7 proteolysis. The perlecan-Sema3A complex abrogates FAK activity and stabilizes PCa cell interactions. MMP-7 expressing cells destroy the complex to initiate metastasis, destroy perlecan-rich borders, and favor invasion and progression to lethal bone disease.