Alan D. Murdoch

Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function.

This review focuses on the extracellular proteoglycans. Special emphasis is placed on the structural features of their protein cores, their gene organization, and their transcriptional control. A simplified nomenclature comprising two broad groups of extracellular proteoglycans is offered: the small leucine‐rich proteoglycans or SLRPs, pronounced “slurps,” and the modular proteoglycans. The first group encompasses at least five distinct members of a gene family characterized by a central domain composed of leucine‐rich repeats flanked by two cysteine‐rich regions. The second group consists of those proteoglycans whose unifying feature is the assembly of various protein modules in a relatively elongated and often highly glycosylated structure. This group is quite heterogeneous and includes a distinct family of proteoglycans, the “hyalectans,” that bind hyaluronan and contain a C‐type lectin motif that is likely to bind carbohydrates, and a less distinct group that contains structural homologies but lacks hyaluronan‐binding properties or lectin‐like domains.—Iozzo, R. V., Murdoch, A. D. Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J. 10, 598‐614 (1996)

Chondrogenic Differentiation of Human Bone Marrow Stem Cells in Transwell Cultures: Generation of Scaffold-Free Cartilage

Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in high‐density cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSCs as a shallow multicellular layer on a permeable membrane over 2–4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6 mm in diameter by 0.8–1 mm in thickness from 0.5 × 106 cells. The discs contained an extensive cartilage‐like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSCs differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage‐specific collagen II, and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors SOX9, L‐SOX5, and SOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold‐free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies.

Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization.

Perlecan, a multidomain heparan sulfate proteoglycan (PG), is an intrinsic component of basement membranes and extracellular matrices. We used a prokaryotic expression vector to generate fusion proteins encoding various domains of human perlecan protein core and these recombinant proteins were used as immunogens to produce mouse anti-human monoclonal antibodies (MAb). One MAb, designated 7B5, was characterized by Western blotting and ELISA and was shown to react specifically with the laminin-like region of perlecan (Domain III) but not with two other fusion proteins encoding Domain II or V. This perlecan epitope was detected by immunoenzymatic staining in the basement membranes of human tissues including pituitary gland, skin, breast, thymus, prostate, colon, liver, pancreas, spleen, heart, and lung. All vascular basement membranes tested contained this gene product. In addition, sinusoidal vessels of liver, spleen, lymph nodes, and pituitary gland expressed high levels of perlecan in the subendothelial region. In situ hybridization, using as probe the same human cDNA-encoding Domain III, localized perlecan mRNA to specific cell types within the tissues and demonstrated that in skin, perlecan appears to be synthesized exclusively by connective tissue cells in the dermal layer. The availability of MAb against precise regions of human perlecan will allow the investigation of this gene product in normal and diseased states.

Tissue engineering: chondrocytes and cartilage

Chapter summary Tissue engineering offers new strategies for developing treatments for the repair and regeneration of damaged and diseased tissues. These treatments, using living cells, will exploit new developments in understanding the principles in cell biology that control and direct cell function. Arthritic diseases that affect so many people and have a major impact on the quality of life provide an important target for tissue engineering. Initial approaches are in cartilage repair; in our own programme we are elucidating the signals required by chondrocytes to promote new matrix assembly. These principles will extend to other tissues of the musculoskeletal system, including the repair of bone, ligament and tendon.

Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells

Perlecan, a major heparan sulfate proteoglycan of vascularized tissues, was immunopurified from media conditioned by human endothelial cells of both arterial and venous origin. The heparan sulfate moiety of perlecan from cultured arterial cells differed in amount and/or composition from that produced by a transformed cell line of venous origin. Both forms of perlecan bound basic fibroblast growth factor with Kd approximately 70 nM. In ELISA experiments, perlecan and its protein core bound to various extracellular matrix components in a manner that was strongly influenced by the format of the assay. Human vascular smooth muscle cells and human endothelial cells adhered to perlecan-coated surfaces, and both cell types adhered better to the venous cell-derived than to the arterial cell-derived perlecan. Removal of the heparan sulfate chains abolished this difference and increased the ability of both types of perlecan to adhere vascular cells. Denaturation of perlecan and its protein core also rendered each of them more adhesive, indicating the presence of conformation-independent adhesion determinants in the polypeptide sequence. Their location was investigated using recombinant perlecan domains. Overall, our results represent the first demonstration of human perlecan acting as an adhesive molecule for human vascular cells and suggest that it may play a role in vascular wound healing.

Structural and functional characterization of the human perlecan gene promoter. Transcriptional activation by transforming growth factor-beta via a nuclear factor 1-binding element.

Perlecan, a modular heparan sulfate proteoglycan of basement membranes and cell surfaces, plays a crucial role in regulating the assembly of extracellular matrices and the binding of nutrients and growth factors to target cells. To achieve a molecular understanding of perlecan gene regulation, we isolated the 5′-flanking region and investigated its functional promoter activity and its response to cytokines. Transient cell transfection assays, using plasmid constructs harboring the perlecan promoter linked to the chloramphenicol acetyltransferase reporter gene, demonstrated that the largest ∼2.5-kilobase construct contained maximal promoter activity. This promoter region was functionally active in a variety of cells of diverse histogenetic origin, thus corroborating the widespread expression of this gene product. Stepwise 5′ deletion analyses demonstrated that the −461-base pair (bp) proximal promoter retained ∼90% of the total activity, and internal deletions confirmed that the most proximal sequence was essential for proper promoter activity. Nanomolar amounts of transforming growth factor-β induced 2-3-fold perlecan mRNA and protein core levels in normal human skin fibroblasts, and this induction was transcriptionally regulated; in contrast, tumor necrosis factor-α had no effect and was incapable of counteracting the effects of TGF-β. Using additional 5′ deletions and DNase footprinting analyses, we mapped the TGF-β responsive region to a sequence of 177 bp contained between −461 and −285. This region harbored a 14-bp element similar to a TGF-β-responsive element present in the promoters of collagen α1(I), α2(I), elastin, and growth hormone. Electrophoretic mobility shift assays and mutational analyses demonstrated that the perlecan TGF-β-responsive element bound specifically to TGF-β-inducible nuclear proteins with high affinity for NF-1 member(s) of transcription factors.

Alternative Splicing in the Aggrecan G3 Domain Influences Binding Interactions with Tenascin-C and Other Extracellular Matrix Proteins

The proteoglycans aggrecan, versican, neurocan, and brevican bind hyaluronan through their N-terminal G1 domains, and other extracellular matrix proteins through the C-type lectin repeat in their C-terminal G3 domains. Here we identify tenascin-C as a ligand for the lectins of all these proteoglycans and map the binding site on the tenascin molecule to fibronectin type III repeats, which corresponds to the proteoglycan lectin-binding site on tenascin-R. In the G3 domain, the C-type lectin is flanked by epidermal growth factor (EGF) repeats and a complement regulatory protein-like motif. In aggrecan, these are subject to alternative splicing. To investigate if these flanking modules affect the C-type lectin ligand interactions, we produced recombinant proteins corresponding to aggrecan G3 splice variants. The G3 variant proteins containing the C-type lectin showed different affinities for various ligands, including tenascin-C, tenascin-R, fibulin-1, and fibulin-2. The presence of an EGF motif enhanced the affinity of interaction, and in particular the splice variant containing both EGF motifs had significantly higher affinity for ligands, such as tenascin-R and fibulin-2. The mRNA for this splice variant was shown by reverse transcriptase-PCR to be expressed in human chondrocytes. Our findings suggest that alternative splicing in the aggrecan G3 domain may be a mechanism for modulating interactions and extracellular matrix assembly.

Cellular methods in cartilage research: Primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells

Work in our laboratory has focused on the in vitro culture of both human articular chondrocytes and human mesenchymal stem cells to understand what controls their ability to synthesise an appropriate cartilage-like extracellular matrix containing a predominantly collagen type II fibrillar network embedded in an aggrecan-rich ECM. This review focuses on the methodologies that we have found to be successful with cartilage and bone marrow sources of human cells and comments on the many factors which may enable improved phenotypic performance once the cells are in a fully chondrogenic environment.

THE PROTEOGLYCAN PERLECAN IS EXPRESSED IN THE ERYTHROLEUKEMIA CELL LINE K562 AND IS UPREGULATED BY SODIUM BUTYRATE AND PHORBOL ESTER

Perlecan is a modular heparan sulfate proteoglycan that harbors five domains with homology to the low density lipoprotein receptor, epidermal growth factor, laminin and neural cell adhesion molecule. Using a monoclonal antibody directed against the laminin-like domain of perlecan, we have recently shown that perlecan is widely expressed in all lymphoreticular systems. To investigate further this observation we have studied the expression of perlecan in two human leukemic cell lines. Using reverse transcriptase-PCR, ribonuclease protection assay, and metabolic labeling we detected significant perlecan expression in the multipotential cell line K562, originally derived from a patient with chronic myelogenous leukemia. In contrast, the promyelocytic cell line HL-60 expressed perlecan at barely detectable levels. These results were intriguing because the K562 cells do not assemble or produce a classical basement membrane. Following induction with either sodium butyrate or the phorbol diester 12-0-tetradecanoylphorbol-13-acetate (TPA), K562 and HL-60 differentiate into early progenitor cells with erythroid or megakaryocytic properties, respectively. Following treatment of K562 and HL-60 cells with either of these agents, perlecan expression was markedly increased in K562 cells. In contrast, we could detect perlecan protein synthesis in HL-60 cells only at very low levels, even after induction with TPA or sodium butyrate. Collectively, these results indicate that perlecan is actively synthesized by bone marrow derived cells and suggest that this proteoglycan may play a role in hematopoietic cell differentiation.

The folded protein modules of the C-terminal G3 domain of aggrecan can each facilitate the translocation and secretion of the extended chondroitin sulfate attachment sequence.

Aggrecan is a multidomain proteoglycan containing both extended and folded protein modules. The C-terminal G3 domain contains a lectin-like, complement regulatory protein-like, and two alternatively spliced epidermal growth factor-like modules. It has been proposed that the lectin module alone has a necessary role in the intracellular translocation and secretion of proteins expressed containing G3. Constructs containing human aggrecan G3 together with 1155 bases of the adjacent chondroitin sulfate attachment region (CS-2) were prepared with different combinations and deletions of the protein modules and transfected into mammalian cells of monkey or hamster origin. The results showed that the products containing only the unfolded protein sequences (CS-2 with or without the C-terminal tail sequence) were translated and accumulated intracellularly but were not secreted. In contrast the constructs containing any of the folded protein modules and the extended CS-2 region were translated and secreted from the cells. The results show that the lectin module was not unique in facilitating the intracellular translocation and secretion of the G3 domain. The conservation of G3-like domains within the aggrecan family of proteoglycans may therefore result from their participation in other extracellular functions.