Perlecan Expression Research Articles

Endothelial Cells Provide Feedback Control for Vascular Remodeling Through a Mechanosensitive Autocrine TGF-β Signaling Pathway

Mechanical forces are potent modulators of the growth and hypertrophy of vascular cells. We examined the molecular mechanisms through which mechanical force and hypertension modulate endothelial cell regulation of vascular homeostasis. Exposure to mechanical strain increased the paracrine inhibition of vascular smooth muscle cells (VSMCs) by endothelial cells. Mechanical strain stimulated the production of perlecan and heparan sulfate glycosaminoglycans by endothelial cells. By inhibiting the expression of perlecan with an antisense vector we demonstrated that perlecan was essential to the strain-mediated effects on endothelial cell growth control. Mechanical regulation of perlecan expression in endothelial cells was governed by a mechanotransduction pathway requiring autocrine transforming growth factor beta (TGF-beta) signaling and intracellular signaling through the ERK pathway. Immunohistochemical staining of the aortae of spontaneously hypertensive rats demonstrated strong correlations between endothelial TGF-beta, phosphorylated signaling intermediates, and arterial thickening. Further, studies on ex vivo arteries exposed to varying levels of pressure demonstrated that ERK and TGF-beta signaling were required for pressure-induced upregulation of endothelial HSPG. Our findings suggest a novel feedback control mechanism in which net arterial remodeling to hemodynamic forces is controlled by a dynamic interplay between growth stimulatory signals from VSMCs and growth inhibitory signals from endothelial cells.

Loss of short dystrophin isoform Dp71 in olfactory ensheathing cells causes vomeronasal nerve defasciculation in mouse olfactory system

The Duchenne muscular dystrophy ( DMD) gene encodes dystrophin, which is a protein defective in DMD patients, as well as a number of shorter isoforms, which have been shown to be expressed in various non-muscle, primarily neural, tissues. As of yet, the physiological function of the various dystrophin isoforms is not fully understood. In the present study, we investigated the neurological phenotype that arises in the DMD-null mice, where expression of all dystrophin isoforms had been disrupted. We demonstrate that vomeronasal axons in the DMD-null mice are defasciculated, and some of the defasciculated vomeronasal axons aberrantly entered into the main olfactory bulb, which indicates that the product(s) of the DMD gene plays an important role in vomeronasal nerve organization. Through western blot and immunofluorescence analyses, we determined that the dystrophin isoform Dp71 was exclusively expressed in the mouse olfactory system: mainly in the olfactory ensheathing cells (OECs), an olfactory system-specific glia cell that ensheaths fascicles of the olfactory nerve. In the OECs, Dp71 was co-localized with β-dystroglycan, utrophin, laminin, and perlecan. Since β-dystroglycan and perlecan expression was decreased in the OECs of DMD-null mice, we hypothesize that Dp71 expressed in the OECs participates in fasciculation of the vomeronasal nerve, most likely through interactions with extracellular matrix.

Sustained VEGF Blockade Results in Microenvironmental Sequestration of VEGF by Tumors and Persistent VEGF Receptor-2 Activation

Vascular endothelial growth factor (VEGF) blockade has been validated clinically as a treatment for human cancers, yet virtually all patients eventually develop progressive disease during therapy. In order to dissect this phenomenon, we examined the effect of sustained VEGF blockade in a model of advanced pediatric cancer. Treatment of late-stage hepatoblastoma xenografts resulted in the initial collapse of the vasculature and significant tumor regression. However, during sustained treatment, vessels recovered, concurrent with a striking increase in tumor expression of perlecan, a heparan sulfate proteoglycan. Whereas VEGF mRNA was expressed at the periphery of surviving clusters of tumor cells, both secreted VEGF and perlecan accumulated circumferential to central vessels. Vascular expression of heparanase, VEGF receptor-2 ligand binding, and receptor activation were concurrently maintained despite circulating unbound VEGF Trap. Endothelial survival signaling via Akt persisted. These findings provide a novel mechanism for vascular survival during sustained VEGF blockade and indicate a role for extracellular matrix molecules that sequester and release biologically active VEGF.

Disruption of Heparan and Chondroitin Sulfate Signaling Enhances Mesenchymal Stem Cell-Derived Osteogenic Differentiation via Bone Morphogenetic Protein Signaling Pathways

Cell surface heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans have been implicated in a multitude of biological processes, including embryonic implantation, tissue morphogenesis, wound repair, and neovascularization through their ability to regulate growth factor activity and morphogenic gradients. However, the direct role of the glycosaminoglycan (GAG) sugar-side chains in the control of human mesenchymal stem cell (hMSC) differentiation into the osteoblast lineage is poorly understood. Here, we show that the abundant cell surface GAGs, HS and CS, are secreted in proteoglycan complexes that directly regulate the bone morphogenetic protein (BMP)-mediated differentiation of hMSCs into osteoblasts. Enzymatic depletion of the HS and CS chains by heparinase and chondroitinase treatment decreased HS and CS expression but did not alter the expression of the HS core proteins perlecan and syndecan. When digested separately, depletion of HS and CS chains did not effect hMSC proliferation but rather increased BMP bioactivity through SMAD1/5/8 intracellular signaling at the same time as increasing canonical Wnt signaling through LEF1 activation. Long-term culturing of cells in HS- and CS-degrading enzymes also increased bone nodule formation, calcium accumulation, and the expression of such osteoblast markers as alkaline phosphatase, RUNX2, and osteocalcin. Thus, the enzymatic disruption of HS and CS chains on cell surface proteoglycans alters BMP and Wnt activity so as to enhance the lineage commitment and osteogenic differentiation of hMSCs.

Perlecan participates in the protection of ischemic myocardium induced by basic fibroblast growth factor: experiment with rats

To investigate if perlecan (PN) is involved in the myocardial protection of basic fibroblast growth factor (bFGF) in acute myocardial infarction. Twenty-four Wistar rats were randomized into 3 groups: myocardial infarction group (MI group, n = 8, undergoing ligation of the descending anterior branch of the left coronary artery), bFGF + MI group (n = 10, injected with bFGF into the border myocardium between the infracted and non- infracted areas immediately after the ligation of the descending anterior branch), and sham operation group (n = 6, undergoing sham operation and injection of normal saline). 24 h, 14 days, and 28 days after the operation the hemodynamic parameters, infarct size, and microvessel density (MVD) were observed. The hearts were taken out, RT-PCR was used to detect the mRNA expression of PN, and Western blotting was used to detect the expression of focal adhesion kinase (FAK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In the bFGF + MI group, the +dp/dt and -dp/dt were improved markedly, the infarct size was significantly smaller, the MVD value was higher than those of the MI group on day 14 and day 28; the perlecan mRNA expression was higher in the infarct marginal area and interior zone; and the expression of FAK and the p38MAPK phosphorylation were up-regulated in the marginal zone of the bFGF group. bFGF is useful in promoting ischemic myocardial angiogenesis; reducing the size of infracted myocardium, and improving the ventricular function in acute myocardial infarction. Perlecan participates in the cardiac protection induced by bFGF. The relevant pathway is related to up-regulation of FAK and p38MAPK.

The Continuous Erythropoietin Receptor Activator Affects Different Pathways of Diabetic Renal Injury

This study explored the tissue-protective properties of the continuous erythropoietin receptor activator (CERA) in an experimental model of (nonischemic) diabetic kidney injury (i.e., the db/db mouse). Mice were randomly treated with placebo (n = 25), low-dosage CERA (n = 25), and high-dosage CERA (n = 25). Also studied were 25 nondiabetic db/m mice. Hematocrit was comparable in placebo and low-dosage CERA-treated mice but increased significantly with high-dosage CERA (P < 0.01 versus both). Significantly reduced expression of TGF-beta, vascular endothelial growth factor, and collagen IV was found in glomeruli and the tubulointerstitial area with CERA treatment, and these beneficial molecular effects were clearly dosage dependent (both P < 0.05 versus placebo). Similarly, CERA treatment caused a dosage-dependent increase in p-Akt, nephrin, and perlecan tissue expression (all P < 0.05 versus placebo). However, the accelerated mesangial expansion that was observed in placebo-treated db/db mice (versus db/m controls) was significantly reduced only in low-dosage CERA-treated mice (P < 0.01). Moreover, albuminuria was significantly reduced in low- but not high-dosage CERA-treated mice compared with placebo treatment (P < 0.05). In an ancillary study, phlebotomy was performed in high-dosage CERA-treated db/db mice to keep hematocrit within normal (baseline) levels. This procedure resulted in significantly (P < 0.05) less albuminuria as compared with high-dosage CERA-treated mice without phlebotomy, thus preserving the tissue-protective potential of CERA. Long-term CERA treatment has beneficial dosage-dependent effects on molecular pathways of diabetic kidney damage. Low-dosage CERA does not affect hematocrit and therefore may be a feasible method of tissue protection in this setting.

Glycosaminoglycans and Proteoglycans: Overlooked Entities?

By virtue of their high net negative charge, glycosaminoglycans and proteoglycans play pivotal roles in biologic processes such as cell-cell and cell-matrix interactions, sequestration of growth factors, activation of chemokines and cytokines, and permselectivity of basement membranes. The present article reviews the putative roles of glycosaminoglycans and proteoglycans in the peritoneal cavity during normal peritoneal homeostasis and chronic inflammation, the latter induced by constant exposure of the peritoneum to non-physiologic peritoneal dialysis (PD) solutions. Glycosaminoglycans have been identified in the mesothelial glycocalyx, a slippery, non-adhesive layer that protects the peritoneal membrane from abrasion and infection. Dermatan sulfate proteoglycans can neutralize the activity of transforming growth factor beta1 and can thus play an essential role in modulating peritoneal fibrosis. Heparan sulfate proteoglycans play a crucial role in the sequestration of growth factors; they also modulate selective permeability of proteins across the peritoneal cavity. Reduced expression of perlecan, a heparan sulfate proteoglycan of the basement membrane, is observed in peritoneal biopsies obtained from established PD patients, consequent to prolonged exposure to the elevated glucose concentrations in conventional PD solutions. Supplementation of PD fluids with glycosaminoglycans has been shown to be beneficial to both the structural and functional integrity of the peritoneum. Recent advances in the field of glycobiology have revealed a multitude of biologic processes that are controlled or influenced by glycosaminoglycans and proteoglycans. Altered synthesis of these macromolecules during PD has serious implications for the peritoneal transport of proteins, host defense, wound healing, inflammation, and fibrosis.

Perlecan, a candidate gene for the CAPB locus, regulates prostate cancer cell growth via the Sonic Hedgehog pathway: Datta MW, Hernandez AM, Schlicht MJ, Kahler AJ, DeGueme AM, Dhir R, Shah RB, Farach-Carson C, Barrett A, Datta S, Department of Pathology, Emory University, Atlanta, GA

Genetic studies associated the CAPB locus with familial risk of brain and prostate cancers. We have identified HSPG2 (Perlecan) as a candidate gene for CAPB. Previously we have linked Perlecan to Hedgehog signaling in Drosophila. More recently, we have demonstrated the importance of Hedgehog signaling in humans for advanced prostate cancer. Here we demonstrate Perlecan expression in prostate cancer, and its function in prostate cancer cell growth through interaction and modulation of Sonic Hedgehog (SHH) signaling. Perlecan expression in prostate cancer tissues correlates with a high Gleason score and rapid cell proliferation. Perlecan is highly expressed in prostate cancer cell lines, including androgen insensitive cell lines and cell lines selected for metastatic properties. Inhibition of Perlecan expression in these cell lines decreases cell growth. Simultaneous blockade of Perlecan expression and androgen signaling in the androgen-sensitive cell line LNCaP was additive, indicating the independence of these two pathways. Perlecan expression correlates with SHH in tumor tissue microarrays and increased tumor cell proliferation based on Ki-67 immunohistochemistry. Inhibition of Perlecan expression by siRNA in prostate cancer cell lines decreases SHH signaling while expression of the downstream SHH effector GLI1 rescues the proliferation defect. Perlecan forms complexes with increasing amounts of SHH that correlate with increasing metastatic potential of the prostate cancer cell line. SHH signaling also increases in the more metastatic cell lines. Metastatic prostate cancer cell lines grown under serum-starved conditions (low androgen and growth factors) resulted in maintenance of Perlecan expression. Under low androgen, low growth factor conditions, Perlecan expression level correlates with the ability of the cells to maintain SHH signaling. We have demonstrated that Perlecan, a candidate gene for the CAPB locus, is a new component of the SHH pathway in prostate tumors and works independently of androgen signaling. In metastatic tumor cells increased SHH signaling correlates with the maintenance of Perlecan expression and more Perlecan-SHH complexes. Perlecan is a proteoglycan that regulates extracellular and stromal accessibility to growth factors such as SHH, thus allowing for the maintenance of SHH signaling under growth factor limiting conditions. This proteoglycan represents an important central regulator of SHH activity and presents an ideal drug target for blocking SHH effects.

Perlecan a multifunctional extracellular proteoglycan scaffold

Perlecan is a large multidomain heparan sulfate proteoglycan of the extracellular matrix. Expression of this proteoglycan changes dynamically during embryo implantation and placentation. Perlecan is expressed by various cells of the embryo including trophectoderm and trophoblast as well as the maternal compartment, including basal lamina underlying uterine epithelia and endothelia and, most dynamically, in developing decidua. Perlecan supports various biological functions, including cell adhesion, growth factor binding, and modulation of apoptosis. Moreover, studies in other systems demonstrate that perlecan expression and activity can be controlled at many levels, including transcription, alternative splicing, and extracellular proteolysis. This review will discuss changes in perlecan expression that occur during embryo implantation and placentation. Furthermore, we propose a model in which perlecan represents an extracellular scaffold protein that supports complex, distinct functions in its full-length form or smaller forms generated by alternative mRNA splicing, extracellular proteolysis, or glycosidase action.

Spectrum ofHSPG2(Perlecan) mutations in patients with Schwartz-Jampel syndrome

Schwartz-Jampel syndrome (SJS) is a rare autosomal recessive condition defined by the association of myotonia with chondrodysplasia. SJS results from mutations in the HSPG2 gene, which encodes perlecan, a major component of basement membranes. Only eight HSPG2 mutations have been reported in six SJS families. Here, we describe the molecular findings in 23 families (35 patients) with SJS, being one-third of the SJS cases reported in the medical literature. We identified 22 new HSPG2 mutations and unreported polymorphisms. Mutations included nine deletion or insertion (41%), six splice site (27%), five missense (23%), and two nonsense mutations (9%). All but four mutations were private, and we found no evidence for a founder effect. Analyses of HSPG2 messenger RNA (mRNA) and perlecan immunostaining on patients' cells revealed a hypomorphic effect of the studied mutations. They also demonstrated distinct consequences of truncating and missense mutations on perlecan expression as truncating mutations resulted in instability of HSPG2 mRNA through nonsense mRNA-mediated decay, whereas missense mutations involving cysteine residues led to intracellular retention of perlecan, probably due to quality control pathways. Our analyses strengthen the idea that SJS results from hypomorphic mutations of the HSPG2 gene. They also propose tools for its molecular diagnosis and provide new clues for the understanding of its pathophysiology.

Glypican-1 Is Frequently Overexpressed in Human Gliomas and Enhances FGF-2 Signaling in Glioma Cells

Signaling by fibroblast growth factor 2 (FGF-2), an autocrine stimulator of glioma growth, is regulated by heparan sulfate proteoglycans (HSPGs) via a ternary complex with FGF-2 and the FGF receptor (FGFR). To characterize glioma growth signaling, we examined whether altered HSPGs contribute to loss of growth control in gliomas. In a screen of five human glioma cell lines, U118 and U251 cell HSPGs activated FGF-2 signaling via FGFR1c. The direct comparison of U251 glioma cells with normal astrocyte HSPGs demonstrated that the glioma HSPGs had a significantly elevated ability to promote FGF-2-dependent mitogenic signaling via FGFR1c. This enhanced activity correlated with a higher level of overall sulfation, specifically the abundance of 2S- and 6S-containing disaccharides. Glioma cell expression of the cell-surface HSPG glypican-1 closely mirrored the FGF-2 coactivator activity. Furthermore, forced expression of glypican-1 in (glypican-1-deficient) U87 glioma cells enhanced their FGF-2 response. Immunohistochemical analysis revealed a highly significant overexpression of glypican-1 in human astrocytoma and oligodendroglioma samples compared with nonneoplastic gliosis. In summary, these observations suggest that altered HSPGs contribute to enhanced signaling of FGF-2 via FGFR1c in gliomas with glypican-1 playing a significant role in this mitogenic pathway.

Characterization of lymphangiogenesis in a model of adult skin regeneration

To date, adult lymphangiogenesis is not well understood. In this study we describe the evolution of lymphatic capillaries in regenerating skin and correlate lymphatic migration and organization with the expression of matrix metalloproteinases (MMPs), immune cells, the growth factors VEGF-A and VEGF-C, and the heparan sulfate proteogylcan perlecan, a key component of basement membrane. We show that while lymphatic endothelial cells (LECs) migrate and organize unidirectionally, in the direction of interstitial fluid flow, they do not sprout into the region but rather migrate as single cells that later join together into vessels. Furthermore, in a modified "shunted flow" version of the model, infiltrated LECs fail to organize into functional vessels, indicating that interstitial fluid flow is necessary for lymphatic organization. Perlecan expression on new lymphatic vessels was only observed after vessel organization was complete and also appeared first in the distal region, consistent with the directionality of lymphatic migration and organization. VEGF-C expression peaked at the initiation of lymphangiogenesis but was reduced to lower levels throughout organization and maturation. In mice lacking MMP-9, lymphatics regenerated normally, suggesting that MMP-9 is not required for lymphangiogenesis, at least in mouse skin. This study thus characterizes the process of adult lymphangiogenesis and differentiates it from sprouting blood angiogenesis, verifies its dependence on interstitial fluid flow for vessel organization, and correlates its temporal evolution with those of relevant environmental factors.

Podocyte proteoglycan synthesis is involved in the development of nephrotic syndrome

Proteoglycans (PGs) are important for the glomerular barrier, for cell signaling, and for the anchorage of cells to the glomerular basement membrane. They are, however, complex macromolecules, and their production has not yet been thoroughly investigated in podocytes. In the present study, we studied the biosynthesis of PGs by highly differentiated human podocytes and in rats. The cells were treated with puromycin aminonucleoside (PAN; a nephrosis-inducing agent), steroids (used as primary treatment for nephrotic syndrome), or both. Analysis was made by TaqMan real-time PCR, Western blotting, and by metabolic labeling with (35)S and (3)H. We found that podocytes produce versican, syndecan-1, decorin, and biglycan together with the previously known PG syndecan-4, glypican, and perlecan. PAN treatment downregulated the mRNA and the protein expression of both versican (by 24 +/- 6%, P < 0.01, for mRNA and by 50% for protein) and perlecan (by 14 +/- 5%, P < 0.05, for mRNA and by 50% for protein). The decreased expression was confirmed by studying the glomerular gene expression in rats treated with PAN during a time course study. In addition, puromycin decreased the expression of enzymes involved in the glycosaminoglycan biosynthesis. Steroid treatment decreased perlecan (by 24 +/- 3%, P < 0.01) and syndecan-1 expression (by 30 +/- 4%, P < 0.01) but increased the expression of decorin 2.5-fold. The observed alterations of PG synthesis induced by PAN may lead to decreased glomerular anionic charge and disturbed podocyte morphology, factors that are important for the development of a nephrotic syndrome.

Reduced perlecan expression and accumulation in human carotid atherosclerotic lesions

Heparan sulfate in the extracellular matrix of the artery wall has been proposed to possess anti-atherogenic properties by interfering with lipoprotein retention, suppression of inflammation, and inhibition of smooth muscle cell growth. Previously, the amount of heparan sulfate in atherosclerotic lesions from humans and animals has been shown to be reduced but the identity or identities of the heparan sulfate molecules being down regulated in this disease are not known. In this study, atherosclerotic lesions were retrieved from 44 patients undergoing surgery for symptomatic carotid stenosis. Normal iliac arteries from organ donors were used as controls. Analysis of the specimens by gene microarray showed a selective reduction in perlecan gene expression, whereas, expression of the other heparan sulfate proteoglycans in the artery wall, agrin and collagen XVIII, remained unchanged. Expression of the large chondroitin sulfate proteoglycan, versican, also remained unchanged. Real-time PCR confirmed the decrease in perlecan gene expression and the unchanged expression of versican. The findings were supported by immunohistochemical analysis demonstrating a reduced accumulation of both perlecan core protein and heparan sulfate in carotid lesions. The study demonstrates a reduction of perlecan mRNA-expression and protein deposition in human atherosclerosis, which in part explains the low levels of heparan sulfate in this disease.

Urinary protein expression patterns in children with sleep-disordered breathing: Preliminary findings

BackgroundObstructive sleep apnea (OSA) is the most common form of sleep-disordered breathing, with almost 15 million Americans affected and many more at risk. Current diagnostic approach to OSA requires polysomnography, which is laborious, onerous, and time-consuming. There is ample evidence that inflammatory responses to the perturbations associated with OSA trigger a variety of genes and signaling cascades that ultimately lead to end-organ injury and changes in kidney function and protein expression. The aim of this study was therefore to analyze proteins in human urine and assess whether differential expression of particular proteins is associated with the presence of OSA. MethodsEleven OSA and 11 control children between the ages of three and 14 (males=17; females=5) underwent overnight sleep studies followed by a first-morning urine sample. Proteomic analysis of urine samples yielded a unique map of proteins, of which, five spots were selected for further analysis due to their significant intensity differences between OSA and control groups. ResultsMass spectrometry followed by peptide mass fingerprinting conclusively identified four of the five spots as gelsolin, perlecan (a heparan sulfate proteoglycan), albumin, and immunoglobulin (P<0.05 and protein scores>67). ConclusionsOverall, increased expression of gelsolin and perlecan in the urinary proteome of children with OSA suggests that the episodic hypoxia associated with OSA may lead to changes in protein permeability or alternatively to increased catabolism of these proteins and their excretion in urine.

Perlecan, a candidate gene for the CAPB locus, regulates prostate cancer cell growth via the Sonic Hedgehog pathway

BackgroundGenetic studies associated the CAPB locus with familial risk of brain and prostate cancers. We have identified HSPG2 (Perlecan) as a candidate gene for CAPB. Previously we have linked Perlecan to Hedgehog signaling in Drosophila. More recently, we have demonstrated the importance of Hedgehog signaling in humans for advanced prostate cancer.ResultsHere we demonstrate Perlecan expression in prostate cancer, and its function in prostate cancer cell growth through interaction and modulation of Sonic Hedgehog (SHH) signaling. Perlecan expression in prostate cancer tissues correlates with a high Gleason score and rapid cell proliferation. Perlecan is highly expressed in prostate cancer cell lines, including androgen insensitive cell lines and cell lines selected for metastatic properties. Inhibition of Perlecan expression in these cell lines decreases cell growth. Simultaneous blockade of Perlecan expression and androgen signaling in the androgen-sensitive cell line LNCaP was additive, indicating the independence of these two pathways. Perlecan expression correlates with SHH in tumor tissue microarrays and increased tumor cell proliferation based on Ki-67 immunohistochemistry. Inhibition of Perlecan expression by siRNA in prostate cancer cell lines decreases SHH signaling while expression of the downstream SHH effector GLI1 rescues the proliferation defect. Perlecan forms complexes with increasing amounts of SHH that correlate with increasing metastatic potential of the prostate cancer cell line. SHH signaling also increases in the more metastatic cell lines. Metastatic prostate cancer cell lines grown under serum-starved conditions (low androgen and growth factors) resulted in maintenance of Perlecan expression. Under low androgen, low growth factor conditions, Perlecan expression level correlates with the ability of the cells to maintain SHH signaling.ConclusionWe have demonstrated that Perlecan, a candidate gene for the CAPB locus, is a new component of the SHH pathway in prostate tumors and works independently of androgen signaling. In metastatic tumor cells increased SHH signaling correlates with the maintenance of Perlecan expression and more Perlecan-SHH complexes. Perlecan is a proteoglycan that regulates extracellular and stromal accessibility to growth factors such as SHH, thus allowing for the maintenance of SHH signaling under growth factor limiting conditions. This proteoglycan represents an important central regulator of SHH activity and presents an ideal drug target for blocking SHH effects.

Targeting Perlecan in Human Keratinocytes Reveals Novel Roles for Perlecan in Epidermal Formation

Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of beta1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis.

Perlecan signaling: Helping hedgehog stimulate prostate cancer growth

Perlecan, an extracellular matrix proteoglycan, regulates signaling by a variety of growth factors through protein–protein and protein–carbohydrate interactions. Recent evidence demonstrates that Perlecan modulates sonic hedgehog signaling during both development and neoplasia, in particular in prostate cancer. Perlecan directly binds to sonic hedgehog and is required for its signaling. Increased sonic hedgehog signaling due to Perlecan in aggressive and metastatic prostate cancer cells can be attributed to increased Perlecan expression or changes in Perlecan glycan structure. Additional co-localization studies suggest that other tumor types may also have a Perlecan-modulated hedgehog signaling pathway. Inhibitors of Perlecan function at either the protein or glycan level would be ideal drug candidates for anti-cancer therapies.

Decorin and biglycan expression is differentially altered in several muscular dystrophies

Biglycan and decorin are small extracellular proteoglycans that interact with cytokines, whose activity they may modulate, and with matrix proteins, particularly collagens. To better understand their role in muscle fibrosis, we investigated expression of decorin and biglycan transcripts and protein in muscle of several forms of muscular dystrophy, and also expression of perlecan, an extracellular proteoglycan unrelated to collagen deposition. In Duchenne muscular dystrophy (DMD) and LAMA2-mutated congenital muscular dystrophy (MDC1A) we also quantitated transcript levels of the profibrotic cytokine TGF-beta1. We examined muscle biopsies from nine DMD patients, aged 2-8 years; 14 BMD (Becker muscular dystrophy) patients (nine aged 1-5 years; five aged 30-37 years); four MDC1A patients (aged 2-7 years); six dysferlin-deficient patients (aged 19-53 years) with mutation ascertained in two, and normal expression of proteins related to limb girdle muscular dystrophies in the others; 10 sarcoglycan-deficient patients: seven with alpha-sarcoglycan mutation, two with beta-sarcoglycan mutation and one with gamma-sarcoglycan mutation (five aged 8-15 years; five aged 26-43 years); and nine children (aged 1-6 years) and 12 adults (aged 16-61 years) suspected of neuromuscular disease, but who had normal muscle on biopsy. Biglycan mRNA levels varied in DMD and MDC1A depending on the quantitation method, but were upregulated in BMD, sarcoglycanopathies and dysferlinopathy. Decorin mRNA was significantly downregulated in DMD and MDC1A, whereas TGF-beta1 was significantly upregulated. Decorin mRNA was normal in paediatric BMD, but upregulated in adult BMD, sarcoglycanopathies and dysferlinopathy. Perlecan transcript levels were similar to those of age-matched controls in all disease groups. By immunohistochemistry, decorin and biglycan were mainly localized in muscle connective tissue; their presence increased in relation to increased fibrosis in all dystrophic muscle. By visual inspection, decorin bands on immunoblot did not differ from those of age-matched controls in all patient groups. However, when the intensity of the bands was quantitated against vimentin and normalized against sarcomeric actin, in DMD and MDC1A the ratio of band intensities was significantly lower than in age-matched controls. Variations in the transcript and protein levels of these proteoglycans in different muscular dystrophies probably reflect the variable disruption of extracellular matrix organization that occurs in these diseases. The significantly lowered decorin levels in DMD and MDC1A may be related to the increased TGF-beta1 levels, suggesting a therapeutic role of decorin in these severe dystrophies.

Altered perlecan expression in placental development and gestational diabetes mellitus

The proteoglycan perlecan is involved in cell signaling, regulation of growth factor activity, and maintenance of basement membranes. This study aims to investigate the expression of perlecan during placental development and whether hyperglycemia of gestational diabetes mellitus induces the alteration of perlecan expression in placenta. Immunohistochemistry, immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and quantitative real-time PCR were carried out to study the placental perlecan expression at different trimesters of pregnancies and in gestational diabetes mellitus. The perlecan protein was mainly immunolocalized in the trophoblast and vessel basement membranes with some staining in the villous stroma of placental villus. Perlecan was also found to co-localize with laminin and collagen IV in the basement membranes of placenta. The protein and mRNA levels of placental perlecan were significantly decreased as the gestational age increased. However, a significant increase in perlecan expression was observed in the third trimester placentas with gestational diabetes mellitus compared to the gestational age-matched controls. Furthermore, trophoblast cells cultured in a high glucose (30 mM) medium and a high osmotic pressure medium (5.6 mM glucose and 24.4 mM mannitol) showed increased perlecan expression compared to cells cultured in the low glucose (5.6 mM) regular medium. These alterations of perlecan expression may be associated with the structural changes of placenta during maturation. The metabolic effect of high glucose and high osmotic pressure of gestational diabetes mellitus may contribute to the increased perlecan expression of diabetic placentas.