Brian J. Grindel

Perlecan/Hspg2 deficiency alters the pericellular space of the lacunocanalicular system surrounding osteocytic processes in cortical bone.

Osteocytes project long, slender processes throughout the mineralized matrix of bone, where they connect and communicate with effector cells. The interconnected cellular projections form the functional lacunocanalicular system, allowing fluid to pass for cell‐to‐cell communication and nutrient and waste exchange. Prevention of mineralization in the pericellular space of the lacunocanalicular pericellular space is crucial for uninhibited interstitial fluid movement. Factors contributing to the ability of the pericellular space of the lacunocanalicular system to remain open and unmineralized are unclear. Immunofluorescence and immunogold localization by transmission electron microscopy demonstrated perlecan/Hspg2 signal localized to the osteocyte lacunocanalicular system of cortical bone, and this proteoglycan was found in the pericellular space of the lacunocanalicular system. In this study we examined osteocyte lacunocanalicular morphology in mice deficient in the large heparan sulfate proteoglycan perlecan/Hspg2 in this tissue. Ultrastructural measurements with electron microscopy of perlecan/Hspg2‐deficient mice demonstrated diminished osteocyte canalicular pericellular area, resulting from a reduction in the total canalicular area. Additionally, perlecan/Hspg2‐deficient mice showed decreased canalicular density and a reduced number of transverse tethering elements per canaliculus. These data indicated that perlecan/Hspg2 contributed to the integrity of the osteocyte lacunocanalicular system by maintaining the size of the pericellular space, an essential task to promote uninhibited interstitial fluid movement in this mechanosensitive environment. This work thus identified a new barrier function for perlecan/Hspg2 in murine cortical bone.

Matrilysin/matrix metalloproteinase-7(MMP7) cleavage of perlecan/HSPG2 creates a molecular switch to alter prostate cancer cell behavior

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.

Tumor necrosis factor-α treatment of HepG2 cells mobilizes a cytoplasmic pool of ERp57/1,25D3-MARRS to the nucleus

ERp57/PDIA3/1,25‐MARRS has diverse functions and multiple cellular locations in various cell types. While classically described as an endoplasmic reticulum (ER) resident protein, ERp57 has a nuclear location sequence (NLS) and can enter the nucleus from the cytosol to alter transcription of target genes. Dysregulation and variable expression of ERp57 is associated with a variety of cancers including hepatocellular carcinoma (HCC). We investigated the dynamic mobility of ERp57 in an HCC cell line, HepG2, to better understand the movement and function of the non‐ER resident pool of ERp57. Subcellular fractionation indicated ERp57 is highly expressed in the ER with a smaller cytoplasmic pool in HepG2 cells. Utilizing an ERp57 green fluorescent protein fusion construct created with and without a secretory signal sequence, we found that cytoplasmic ERp57 translocated to the nucleus within 15 min after tumor necrosis factor‐α (TNF‐α) treatment. Protein kinase C activators including 1,25‐dihydroxyvitamin D3 and phorbol myristate acetate did not trigger nuclear translocation of ERp57, indicating translocation is PKC independent. To determine if an interaction between the rel homology binding domain in ERp57 and the nuclear factor‐κB subunit, p65, occurred after TNF‐α treatment and could account for nuclear movement, co‐immunoprecipitation was performed under control and conditions that stabilized labile disulfide bonds. No support for a functional interaction between p65 and ERp57 after TNF‐α treatment was found in either case. Immunostaining for both ERp57‐GFP and p65 after TNF‐α treatment indicated that nuclear translocation of these two proteins occurs independently in HepG2 cells. J. Cell. Biochem. 112: 2606–2615, 2011.

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 bones 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 Youngs 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.

Transcriptional activation by NFκB increases perlecan/HSPG2 expression in the desmoplastic prostate tumor microenvironment.

Perlecan/HSPG2, a heparan sulfate proteoglycan typically found at tissue borders including those separating epithelia and connective tissue, increases near sites of invasion of primary prostatic tumors as previously shown for other proteins involved in desmoplastic tissue reaction. Studies of prostate cancer cells and stromal cells from both prostate and bone, the major site for prostate cancer metastasis, showed that cancer cells and a subset of stromal cells increased production of perlecan in response to cytokines present in the tumor microenvironment. In silico analysis of the HSPG2 promoter revealed two conserved NFκB binding sites, in addition to the previously reported SMAD3 binding sites. By systematically transfecting cells with a variety of reporter constructs including sequences up to 2.6 kb from the start site of transcription, we identified an active cis element in the distal region of the HSPG2 promoter, and showed that it functions in regulating transcription of HSPG2. Treatment with TNF‐α and/or TGFβ1 identified TNF‐α as a major cytokine regulator of perlecan production. TNF‐α treatment also triggered p65 nuclear translocation and binding to the HSPG2 regulatory region in stromal cells and cancer cells. In addition to stromal induction of perlecan production in the prostate, we identified a matrix‐secreting bone marrow stromal cell type that may represent the source for increases in perlecan in the metastatic bone marrow environment. These studies implicate perlecan in cytokine‐mediated, innate tissue responses to cancer cell invasion, a process we suggest reflects a modified wound healing tissue response co‐opted by prostate cancer cells. J. Cell. Biochem. 115: 1322–1333, 2014.

Perlecan/HSPG2 and matrilysin/MMP-7 as indices of tissue invasion: tissue localization and circulating perlecan fragments in a cohort of 288 radical prostatectomy patients.

Prostate cancer (PCa) cells use matrix metalloproteinases (MMPs) to degrade tissue during invasion. Perlecan/HSPG2 is degraded at basement membranes, in reactive stroma and in bone marrow during metastasis. We previously showed MMP-7 efficiently degrades perlecan. We now analyzed PCa tissue and serum from 288 prostatectomy patients of various Gleason grades to decipher the relationship between perlecan and MMP-7 in invasive PCa. In 157 prostatectomy specimens examined by tissue microarray, perlecan levels were 18% higher than their normal counterparts. In Gleason grade 4 tissues, MMP-7 and perlecan immunostaining levels were highly correlated with each other (average correlation coefficient of 0.52) in PCa tissue, regardless of grade. Serial sections showed intense, but non-overlapping, immunostaining for MMP-7 and perlecan at adjacent borders, reflecting the protease-substrate relationship. Using a capture assay, analysis of 288 PCa sera collected at prostatectomy showed elevated levels of perlecan fragments, with most derived from domain IV. Perlecan fragments in PCa sera were associated with overall MMP-7 staining levels in PCa tissues. Domain IV perlecan fragments were present in stage IV, but absent in normal, sera, suggesting perlecan degradation during metastasis. Together, perlecan fragments in sera and MMP-7 in tissues of PCa patients are measures of invasive PCa.

Chaperone Properties of Pdia3 Participate in Rapid Membrane Actions of 1α,25-Dihydroxyvitamin D3

Protein disulfide isomerase family A, member 3 (Pdia3) mediates many of the plasma membrane (PM)-associated rapid responses to 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3). It is not well understood how Pdia3, which is an endoplasmic reticulum (ER) chaperone, functions as a PM receptor for 1α,25(OH)2D3. We mutated 3 amino acids (K214 and R282 in the calreticulin interaction site and C406 in the isomerase catalytic site), which are important for Pdia3s ER chaperone function, and examined their role in responses to 1α,25(OH)2D3. Pdia3 constructs with and without the ER retention signal KDEL were used to investigate the PM requirement for Pdia3. Finally, we determined whether palmitoylation and/or myristoylation were required for Pdia3-mediated responses to 1α,25(OH)2D3. Overexpressing the Pdia3 R282A mutant in MC3T3-E1 cells increased PM phospholipase A2-activating protein, Rous sarcoma oncogene (c-Src), and caveolin-1 but blocked increases in 1α,25(OH)2D3-stimulated protein kinase C (PKC) seen in cells overexpressing wild-type Pdia3 (Pdia3Ovr cells). Cells overexpressing Pdia3 with K214A and C406S mutations had PKC activity comparable to untreated controls, indicating that the native response to 1α,25(OH)2D3 also was blocked. Overexpressing Pdia3[-KDEL] increased PM localization and augmented baseline PKC, but the stimulatory effect of 1α,25(OH)2D3 was comparable to that seen in wild-type cultures. In contrast, 1α,25(OH)2D3 increased prostaglandin E2 in Pdia3[±KDEL] cells. Although neither palmitoylation nor myristoylation was required for PM association of Pdia3, myristoylation was needed for PKC activation. These data indicate that both the chaperone functional domains and the subcellular location of Pdia3 control rapid membrane responses to 1α,25(OH)2D3.

Sustained delivery of recombinant human bone morphogenetic protein-2 from perlecan domain I - functionalized electrospun poly (ε-caprolactone) scaffolds for bone regeneration

BackgroundBiomaterial scaffolds that deliver growth factors such as recombinant human bone morphogenetic proteins-2 (rhBMP-2) have improved clinical bone tissue engineering by enhancing bone tissue regeneration. This approach could be further improved if the controlled delivery of bioactive rhBMP-2 were sustained throughout the duration of osteogenesis from fibrous scaffolds that provide control over dose and bioactivity of rhBMP-2. In nature, heparan sulfate attached to core proteoglycans serves as the co-receptor that delivers growth factors to support tissue morphogenesis.MethodsTo mimic this behavior, we conjugated heparan sulfate decorated recombinant domain I of perlecan/HSPG2 onto an electrospun poly(ε-caprolactone) (PCL) scaffold, hypothesizing that the heparan sulfate chains will enhance rhBMP-2 loading onto the scaffold and preserve delivered rhBMP-2 bioactivity.ResultsIn this study, we demonstrated that covalently conjugated perlecan domain I increased loading capacity of rhBMP-2 onto PCL scaffolds when compared to control unconjugated scaffolds. Additionally, rhBMP-2 released from the modified scaffolds enhanced alkaline phosphatase activity in W20–17 mouse bone marrow stromal cells, indicating the preservation of rhBMP-2 bioactivity indicative of osteogenesis.ConclusionsWe conclude that this platform provides a sophisticated and efficient approach to deliver bioactive rhBMP-2 for bone tissue regeneration applications.

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

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.

Abstract 5063: Matrilysin cleavage of perlecan produces bioactive fragments acting as a molecular switch to alter prostate cancer cell behavior.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Perlecan/HSPG2[PLN], a large heparan sulfate (HS) proteoglycan, 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 PLN. After proteolysis, fragments of PLN can be released into circulation. A sandwich ELISA with antibodies generated across PLN detected fragments of PLN from IG-repeat domain IV in PCa patient serum. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for PLN proteolysis both in silico and in vitro. Purified PLN showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when embedded with other BM proteins. MMP-7 fragments of PLN were detected as limit peptides by western blot. While not predicted in silico, MMP-7 cleaved every subpart of recombinantly generated PLN domain IV. The resultant MMP-7 generated PLN Dm IV fragments were identified by mass spectrometry (LC-MS/MS and MALDI) and Edman degradation N-terminal sequencing. Other PCa relevant enzymes had limited ability to cleave PLN including prostate specific antigen, hepsin and fibroblast activation protein. To determine if PLN digestion affects metastatic PCa cells, C4-2B cells were seeded on plates coated with different forms of PLN. Cells on intact PLN formed large clusters of poorly adherent cells, while those on PLN digested with MMP-7 or heparitinase to remove the HS chains attached and spread within 30 minutes. Immunohistochemistry of PCa patient tissue indicates MMP-7 and PLN are in direct contact at interfaces in the stroma. We conclude that enzymatic processing of PLN in the BM and stroma as occurs in the tumor microenvironment can alter PCa cell behavior and favor cell dispersion and invasiveness. Citation Format: Brian J. Grindel. Matrilysin cleavage of perlecan produces bioactive fragments acting as a molecular switch to alter prostate cancer cell behavior. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5063. doi:10.1158/1538-7445.AM2013-5063