Perlecan/HSPG2: Signaling role of domain IV in chondrocyte clustering with implications for Schwartz-Jampel Syndrome.

Perlecan/heparan sulfate proteoglycan 2 (HSPG2), a large HSPG, is indispensable for the development of musculoskeletal tissues, where it is deposited within the pericellular matrix (PCM) surrounding chondrocytes and disappears nearly completely at the chondro-osseous junction (COJ) of developing long bones. Destruction of perlecan at the COJ converts an avascular cartilage compartment into one that permits blood vessel infiltration and osteogenesis. Mutations in perlecan are associated with chondrodysplasia with widespread musculoskeletal and joint defects. This study elucidated novel signaling roles of perlecan core protein in endochondral bone formation and chondrocyte behavior. Perlecan subdomains were tested for chondrogenic properties in ATDC5 cells, a model for early chondrogenesis. A region within domain IV of perlecan (HSPG2 IV-3) was found to promote rapid prechondrocyte clustering. Introduction of the mutation (R3452Q) associated with the human skeletal disorder Schwartz-Jampel syndrome limited HSPG2 IV-3-induced clustering. HSPG2 IV-3 activity was enhanced when thermally unfolded, likely because of increased exposure of the active motif(s). HSPG2 IV-3-induced clustering was accompanied by the deactivation of key components of the focal adhesion complex, FAK and Src, with increased messenger RNA (mRNA) levels of precartilage condensation markers Sox9 and N-cadherin ( Cdh2), and cartilage PCM components collagen II ( Col2a1) and aggrecan ( Acan). HSPG2 IV-3 reduced signaling through the ERK pathway, where loss of ERK1/2 phosphorylation coincided with reduced FoxM1 protein levels and increased mRNA levels cyclin-dependent kinase inhibitor 1C (Cdkn1c) and activating transcription factor 3 ( Atf3), reducing cell proliferation. These findings point to a critical role for perlecan domain IV in cartilage development through triggering chondrocyte condensation.

Identifying small molecules for protecting chondrocyte function and matrix integrity after controlled compressive injury.

Objective: Articular cartilage injury is central for the development of post-traumatic osteoarthritis (PTOA). With few disease-modifying therapies successful at offsetting progressive osteoarthritis (OA), our goal is to use a high throughput screening platform of cartilage injury to identify novel chondroprotective compounds. Targeting articular cartilage damage immediately after injury remains a promising therapeutic strategy to overcome irreversible tissue damage. Method: We constructed a single impact-cartilage screening method using a multi-platen system that simultaneously impacts 48 samples and makes use of engineered cartilage tissue analogs (known as CTAs). Drug libraries were screened and assessed for their ability to alter two crucial biological responses to impact injuries, namely matrix degradation and cell stress. Results: Over 500 small molecules were screened for their ability to alter proteoglycan loss, matrix metalloproteinase activity, and cell stress or death. Fifty-five compounds passed through secondary screening and were from commercial libraries of natural and redox, stem cell related compounds, as well as protease, kinase and phosphatase inhibitors. Through secondary screening, 16 promising candidates exhibited activity on one or more critical function of chondrocytes. While many are mechanistically known compounds, their function in joint diseases is not known. Conclusion: This platform was validated for screening drug activity against a tissue engineered model of PTOA. Multiple compounds identified in this manner have potential application as early protective therapy for treating PTOA, and require further study. We propose this screening platform can identify novel molecules that act on early chondrocyte responses to injury and provide an invaluable tool for therapeutic development.

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.

Trabecular Bone Deficit and Enhanced Anabolic Response to Re-Ambulation after Disuse in Perlecan-Deficient Skeleton.

Perlecan/Hspg2, a large monomeric heparan sulfate proteoglycan, is found in the basement membrane and extracellular matrix, where it acts as a matrix scaffold, growth factor depot, and tissue barrier. Perlecan deficiency leads to skeletal dysplasia in Schwartz-Jampel Syndrome (SJS) and is a risk factor for osteoporosis. In the SJS-mimicking murine model (Hypo), inferior cortical bone quality and impaired mechanotransduction in osteocytes were reported. This study focused on trabecular bone, where perlecan deficiency was hypothesized to result in structural deficit and altered response to disuse and re-loading. We compared the Hypo versus WT trabecular bone in both axial and appendicular skeletons of 8-38-week-old male mice, and observed severe trabecular deficit in Hypo mice, approximately 50% reduction of Tb.BV/TV regardless of skeletal site and animal age. Defects in endochondral ossification (e.g., accelerated mineralization), increases in osteoclast activity, and altered differentiation of bone progenitor cells in marrow contributed to the Hypo phenotype. The Hypo trabecular bone deteriorated further under three-week hindlimb suspension as did the WT. Re-ambulation partially recovered the lost trabecular bone in Hypo, but not in WT mice. The novel finding that low-impact loading could counter detrimental disuse effects in the perlecan-deficient skeleton suggests a strategy to maintain skeletal health in SJS patients.

Modular Proteoglycan Perlecan/HSPG2: 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'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.

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.

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.