Single molecule force measurements of perlecan/HSPG2: A key component of the osteocyte pericellular matrix

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 that a reduction in perlecan secretion alters the PCM fiber composition and interferes with bone's response to a 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 that perlecan has physical properties that would allow it to act as a strong but elastic tether in the LCS

An Investigation of the Biophysical and Biochemical Properties of Perlecan/HSPG2: Implications for Bone Mechanotransduction and Endochondral Ossification

Perlecan, also known as heparan sulfate proteoglycan 2 (HSPG2), is a very large secreted proteoglycan ubiquitously expressed in all basement membranes and in the territorial matrix of skeletal tissues. Perlecan is particularly important for the formation and stabilization of tissue layers and its absence is catastrophic for bone and cartilage development. In mineralized bone, perlecan is a key component of pericellular matrix (PCM) surrounding osteocytic processes that preserve fluid flow throughout bone tissue. In addition, recent findings have coupled perlecan expression, along the osteocyte cell surface, to bone mechano-adaptive response. Perlecan’s mechanical properties, important for maintaining the osteocyte cell-bone matrix interface, were studied for the first time. This work demonstrated perlecan functions as a semi-flexible tether that is capable of withstanding physiological loads imposed on cortical bone.The second part of this study examined perlecan in the context of early precartilage condensation. This study demonstrated a novel sub-domain of perlecan, PLN IV-3, located in its fourth domain modulates cell-matrix interactions that are involved in the chondrogenic process. When presented as part of the substratum, PLN IV-3 suppressed focal adhesion kinase (FAK) phosphorylation and signaling through the mitogen-activated protein kinase (MAPK) cascade. This led to reduced cell migration, increased cell-cell adhesion, and decreased cell proliferation, all of which are hallmarks for mesenchyme condensation and subsequent precartilage formation. I hypothesize that this is a possible mechanism by which perlecan functions to coordinate the migration of mesodermal cells and drive chondrocyte differentiation as seen during development. Together, these studies suggest perlecan possesses multifunctional properties that affect bone health throughout various stages of life

Modular Proteoglycan Perlecan/<i>HSPG2</i>: Mutations, Phenotypes, and Functions

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&#8217;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

Osteocytic Pericellular Matrix (PCM): Accelerated Degradation under In Vivo Loading and Unloading Conditions Using a Novel Imaging Approach

The proteoglycan-containing pericellular matrix (PCM) controls both the biophysical and biochemical microenvironment of osteocytes, which are the most abundant cells embedded and dispersed in bones. As a molecular sieve, osteocytic PCMs not only regulate mass transport to and from osteocytes but also act as sensors of external mechanical environments. The turnover of osteocytic PCM remains largely unknown due to technical challenges. Here, we report a novel imaging technique based on metabolic labeling and “click-chemistry,” which labels de novo PCM as “halos” surrounding osteocytes in vitro and in vivo. We then tested the method and showed different labeling patterns in young vs. old bones. Further “pulse-chase” experiments revealed dramatic difference in the “half-life” of PCM of cultured osteocytes (~70 h) and that of osteocytes in vivo (~75 d). When mice were subjected to either 3-week hindlimb unloading or 7-week tibial loading (5.1 N, 4 Hz, 3 d/week), PCM half-life was shortened (~20 d) and degradation accelerated. Matrix metallopeptidase MMP-14 was elevated in mechanically loaded osteocytes, which may contribute to PCM degradation. This study provides a detailed procedure that enables semi-quantitative study of the osteocytic PCM remodeling in vivo and in vitro

Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells

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

Perlecan is expressed in the ectoderm of the scapus, irregularly in the mesentery, and irregularly in the inner cell layer of the tentacles.

<p>An adult <i>N</i>. <i>vectensis</i> (A) was tested for <i>perl</i> mRNA expression. The ectoderm of the body wall has consistent <i>perl</i> mRNA signal in the ectoderm, whereas the gastroderm is mostly negative for <i>perl</i> mRNA. The acellular mesoglea is clearly visible in the physus and tentacle (arrowheads, B, D). The mesentery expresses <i>perl</i> mRNA in a sporadic fashion, with pigmented cells not expressing <i>perl</i> (C). Tentacle cells only express <i>perl</i> sporadically in the gastroderm (arrows, D,E). The oral end of the scapus does not express the <i>perl</i> transcript (A, arrowhead, E). Scale bars, A—0.5mm, B-E—40 μm.</p

Comparison of <i>H</i>. <i>sapiens</i> perlecan domain IV and <i>T</i>. <i>adhaerens</i> perl 1 and perl 2 domain IV.

<p>The primary structure of domain IV Ig modules were aligned, including all sequences between disulfide-bonded cysteines in each Ig module. The sequences were aligned and residues conserved in 25% or more of these Ig sequences were highlighted. The central Ig modules of <i>H</i>. <i>sapiens</i> domain IV, distinct from the outer Ig modules, are shown separately. Residues of the central Ig modules that are conserved amongst <i>H</i>. <i>sapiens</i> domain IV Ig modules, but not when compared alongside those of <i>T</i>. <i>adhaerens</i>, are outlined with a red dashed line. Ig sequences originating from <i>T</i>. <i>adhaerens</i> perl 1 and perl 2 are shown separately.</p

Molecular phylogenetic analysis by maximum likelihood method.

<p>The evolutionary history of perlecan was inferred by using the Maximum Likelihood method. Branch lengths are calculated according to nucleic acid substitutions per site. The eumetazoans are delineated by a dotted line.</p

Perlecan is expressed in the regenerating oral region and tentacles at stage 2.5 through stage 4.

<p>Animals were bisected midway through the mesentery region. Representative animals are pictured 48 hours post-bisection in (physal end-A, oral end-B). Regeneration of the oral structures and tentacles was followed in the area of the red box in (A). <i>Perl</i> activation is visible during regenerative stage 2.5–3 in the tentacle bud and oral structures (C). The developing cellular boundary between ectoderm and gastroderm is visible in the primordial tentacle (arrowhead, D) and is distinct from the developed mesoglea in the regenerated oral pole of the scapus (arrow, D). <i>Perl</i> remains elevated in the tentacles and oral structures during the intermediate stage of tentacle regeneration (stage 4, E, F). <i>Perl</i> expression recedes in the tentacles as they develop mesoglea (arrowheads, F) and remains where the mesoglea is not yet fully visible (F, arrows). Prior to completion of regeneration, <i>perl</i> expression continues in the oral ectoderm (asterisks, F). Scale bars, A,B-500μm C,E-100 μm D,F-40μm.</p

Predicted perlecan orthologues or constituents in model organisms.

<p>The presumptive pre-perlecan constituents of <i>A</i>. <i>queenslandica</i> (A) and perlecan proteins of <i>T</i>. <i>adhaerens</i>, <i>N</i>. <i>vectensis</i>, <i>C</i>. <i>elegans</i>, and <i>H</i>. <i>sapiens</i> are shown in schematic form to demonstrate structural differences. Several of the scarce matches for perlecan modules in the <i>M</i>. <i>brevicollis</i> and <i>A</i>. <i>queenslandica</i> genomes are shown in schematic form. The number of unique matches for individual or paired perlecan folding modules in the <i>M</i>. <i>leidyi</i> transcriptome is shown in subscript next to each schematic module. The double line between <i>T</i>. <i>adhaerens perl 1</i> and <i>T</i>. <i>adhaerens perl 2</i> indicates that these two genes, although only thirteen kb apart on the chromosome, are separately transcribed. Blue rectangles represent parts of the <i>T</i>. <i>adhaerens</i> and <i>N</i>. <i>vectensis</i> transcripts amplified by RACE PCR and sequenced; all other parts of the protein were predicted by tBLASTn-alignment. Schematics are not to scale; gene lengths are denoted at the right end of each diagram. The red dashed line encompasses the “perlecan” clade of the animal kingdom.Surprisingly, the easily recognizable <i>perl</i> gene was found in the placozoan <i>T</i>. <i>adhaerens</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124578#pone.0124578.g001" target="_blank">Fig 1</a>). All five protein domains are included in the <i>T</i>. <i>adhaerens perl</i> gene, in the identical order to that of <i>HSPG2</i> in humans. <i>T</i>. <i>adhaerens perl</i> domain I includes a SEA module, which, until now, was thought to be found exclusively in perlecan of mammals and birds. The <i>T</i>. <i>adhaerens perl</i> SEA module includes the G-SVV motif, critical to the autocleavage of these modules in other proteins. Whereas the SEA module of perlecan in mammals and birds does not contain this autocleavage motif, we speculate that this ancestral perlecan molecule may have undergone autocleavage while in the secretory pathway. <i>T</i>. <i>adhaerens perl</i> domain II is structured exactly as that of human <i>HSPG2</i>, with three LDL receptor-like repeats followed by one immunoglobulin (Ig) domain. Domain III of <i>T</i>. <i>adhaerens</i> perl is also similar to that of <i>H</i>. <i>sapiens</i> perlecan/<i>HSPG2</i>, however in comparison, <i>T</i>. <i>adhaerens</i> perl includes one additional set of Laminin B- and Laminin EGF-like modules. Domain IV of <i>T</i>. <i>adhaerens</i> perl encodes roughly 36 total Ig modules, making it substantially larger than the domain IV of human perlecan, which only includes 21 Ig modules. Domain V of <i>T</i>. <i>adhaerens</i> perl encodes both the laminin G and EGF-like modules similar to other perlecan proteins.</p