Vincent C. Hascall

Insulin-like growth factors maintain steady-state metabolism of proteoglycans in bovine articular cartilage explants

The influences of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) on biosynthesis and catabolism of proteoglycans (PG) in bovine articular cartilage explants were examined to define their potential use in a chemically defined medium. In both short- (10 days) and long-term (40 days) cultures, 10 to 20 ng/ml IGF-I maintained PG synthesis at the same or higher levels than in a medium containing 20% fetal calf serum (FCS). Catabolic rates were slower in IGF-I medium than in medium with only 0.1% albumin, but somewhat faster than for cultures in medium with 20% FCS. In long-term cultures 20 ng/ml IGF-I maintained a steady-state condition; the amounts of glycosaminoglycan and DNA per hydroxyproline content were constant throughout the culture period. The half-maximal dose response for IGF-I on PG synthesis (4.5 ng/ml) was distinctly different from that for the IGF-I effect on PG catabolism (1.5 ng/ml), indicating that these two components of PG metabolism can be experimentally uncoupled. IGF-II was less potent than IGF-I in the same batches of articular cartilage; 100 ng/ml IGF-II increased PG synthesis and decreased PG catabolism relative to 0.1% albumin alone, but the responses were only about 60% of those for 5 ng/ml IGF-I. These results suggest that the chondrocytes regulate PG synthesis primarily via the type I IGF receptor and that the IGF-II response is through the same receptor. Evidence is also provided indicating that the cartilage explants initially contain about 50 ng IGF-I per gram wet weight; this matrix-bound IGF-I diffuses into the medium during culture. The chondrocytes synthesize little or no IGF-I that is released into the medium under the culture conditions used.

Mouse oocytes regulate hyaluronic acid synthesis and mucification by FSH-stimulated cumulus cells

Mucification (or expansion) of the cumulus cells surrounding the oocyte is thought to depend on the direct action of gonadotropins in stimulating production and deposition of hyaluronic acid (HA) in the extracellular matrix. We now report that the oocyte is essential for this process. Either follicle-stimulating hormone (FSH) at 1 micrograms/ml or dibutyryl cAMP at 2 mM induces mucification of intact cumulus cell-oocyte complexes (COCs) in vitro, but fails to stimulate mucification of isolated cumulus cells. HA synthesis by FSH-stimulated cumulus cells is only approximately 3.5% of the value achieved by FSH-stimulated COCs. Isolated oocytes cultured with or without FSH do not synthesize detectable amounts of HA but induce isolated cumulus cells to increase HA synthesis approximately 13-fold in cocultures with FSH. Medium conditioned by isolated oocytes for 5 hr induces nearly the same level of HA synthesis by cumulus cells under the same culture conditions. FSH also stimulates cumulus cells to increase synthesis of dermatan sulfate proteoglycans (DS-PGs) approximately 3-fold, but this stimulation does not depend upon the presence of oocytes. The results indicate that oocytes produce a soluble factor(s) essential in combination with FSH to stimulate HA, but not DS-PG, synthesis by cumulus cells in vitro and that this factor(s) acts independently or downstream from the FSH-induced formation of cAMP.

[45] Proteoglycans: Isolation and Characterization

Publisher Summary This chapter presents procedure for extraction and characterization of proteoglycans. In general, tissue or cultures should be processed for extraction quickly and using procedures that minimize endogenous degradation. Fresh tissue should be chilled on ice, cleaned, and minced quickly. Extraction efficiency is often improved when finer tissue pieces are used to expose more surface area to the solvent. Once the proteoglycans are in solution, different fractionation procedures (ion exchange in 7 M urea, rate zonal-velocity, centrifugation molecular-sieve chromatography, electrophoresis in composite polyacrylamide-agarose gels), which take advantage of their macromolecular properties, are used to purify them from other macromolecules in the extract and to begin to separate them from each other. Application of the various techniques described above has identified the different classes of proteoglycans in a starting extract based upon differences in charge density, hydrodynamic size, and buoyant density. Such analyses not only provide information about the physical properties of the proteoglycans but help design effective strategies for purifying at least some of the subpopulations of proteoglycans identified.

The effect of serum on biosynthesis of proteoglycans by bovine articular cartilage in culture

Proteoglycan synthesis by slices of adult bovine articular cartilage is stimulated two-to threefold when tissue is cultured in the presence of fetal calf serum for 5-6 days. After this, essentially steady-state conditions are achieved for up to 14 days in which the high synthetic rates are maintained and the amount of proteoglycan in the tissue remains nearly constant. In the absence of fetal calf serum, synthesis declines to a lower level and there is a gradual, net loss of proteoglycan from the tissue. Tissue maintained without serum for several days rapidly increases synthetic rates to the higher levels over 2-3 days after transferring into medium with serum, and vice versa, indicating that the response of the chondrocytes to serum factors is reversible. The structures of the proteoglycans synthesized under all medium conditions were typical for cartilage. Only small differences in glycosaminoglycan chain sizes and a consistent decrease in the relative amount of keratan sulfate to chondroitin sulfate during the first days in the culture were observed. The net capacity of the cells for chondroitin sulfate synthesis, as estimated by incubation in the presence of exogenous beta-xyloside acceptor, increased (or decreased) in parallel with the changes in endogenous proteoglycan synthesis when cultures were transferred from medium without to medium with serum (or vice versa), suggesting that changes in the net amounts of the enzymes for chondroitin sulfate synthesis are closely coordinated with changes in the amount of core protein being processed to proteoglycans. The responses of calf articular cartilage in the same system were somewhat different. Serum in the medium was required to maintain initial high levels of synthesis. The proteoglycans synthesized contained a lower proportion of keratan sulfate than those initially synthesized in the adult tissue, and there was no change in this proportion with time in culture. The maintenance of steady-state conditions for proteoglycan metabolism by either adult or calf tissue in the presence of serum in these cultures should provide a useful model for studying the regulation of synthesis and catabolism of proteoglycans by chondrocytes residing in a nearly normal extracellular matrix for long periods of time.

Isolation and characterization of proteoglycans.

Publisher Summary Cell culture and tissue explant systems are frequently used as models for investigating proteoglycans. In these cases, radiolabeling methods provide the most convenient ways to follow proteoglycans through purification steps and to monitor recoveries at each step. Sulfate is usually used to label proteoglycans selectively, because more than 90% of the incorporated activity with this precursor will usually be in proteoglycans. The cell layer or tissue usually contains several different proteoglycans. Some can be integrated in the extracellular matrix by noncovalent interactions with other matrix molecules such as collagen. Others can be associated with the cell surface by hydrophobic interaction with the plasma membrane through polypeptide intercalation or phosphatidylinositol anchors, or by ionic binding with other cell surface molecules. Others can be sequestered in intracellular compartments, such as in storage or secretory granules, or in prelysosomal or endosome compartments. Because proteoglycans are easily degraded by proteases, inhibitors against different classes of proteases are usually added to the extraction solvent, and the extractions are usually done at 4°. The protease inhibitors are particularly important in steps in which the solvent is changed to conditions that favor renaturation before the proteoglycans are fully purified because proteases in the extracts can recover activity.

Turnover of proteoglycans in cultures of bovine articular cartilage

Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.

Separate effects of exogenous hyaluronic acid on proteoglycan synthesis and deposition in pericellular matrix by cultured chick embryo limb chondrocytes

Abstract The effects of exogenous hyaluronic acid on cell cultures of chick embryo limb chondrocytes are reported in this paper. The evidence shows that exogenous hyaluronic acid (HA) can both depress the incorporation of 35 SO 4 into glycosaminoglycans and cause a displacement of newly synthesized proteoglycan from the cell layer to the culture medium. The results demonstrate that these two effects are mediated by distinct mechanisms. The displacement effect has a rapid onset (by 2 hr) while the effect of exogenous HA on 35 SO 4 incorporation has a long latency (12 hr). The displacement effect is produced by a lower concentration (5 μg/ml) of hyaluronate oligomers than the effect on 35 SO 4 incorporation (50 μg/ml). In addition, displacement is produced only by hyaluronate oligomers that are decasaccharides or larger. The depression of 35 SO 4 incorporation is produced by tetrasaccharides as well as high molecular weight HA. In fact tetrasaccharides can depress 35 SO 4 incorporation without causing the displacement effect.

Tunicamycin-induced alterations in the synthesis of sulfated proteoglycans and cell surface morphology in the chick embryo fibroblast

Abstract The effect of tunicamycin (TM) on the synthesis and secretion of sulfated proteoglycans and hyaluronate was examined in chick embryo fibroblasts and chondrocytes. The incorporation of the precursors [ 3 H]glucosamine, [ 3 H]mannose and [ 35 S]sulfate into glycoconjugates in both the cell layer and medium of cultures was determined. In the chick embryo fibroblast, but not in the chondrocyte, synthesis of sulfated proteoglycan was inhibited 60–75% by TM (5 × 10 −8 M), while synthesis of hyaluronate and protein was only inhibited slightly. The inhibition of sulfate incorporation into glycosaminoglycans of the chick embryo fibroblast was overcome to a great extent by addition of β-xyloside, which provides an exogenous initiator for chondroitin sulfate synthesis. TM treatment also altered cell shape and surface morphology in chick embryo fibroblasts, as observed by phase contrast and scanning electron microscopy (SEM). Cells treated with TM became rounded, and increased numbers of microvilli and blebs appeared on the cell surface. These alterations in cell morphology were reversed by removal of TM, but not by exogenous addition of xyloside, chondroitin sulfate or the adhesive cell surface glycoprotein fibronectin. These results demonstrate that TM inhibits synthesis of sulfated proteoglycans in the chick embryo fibroblast and causes a dramatic alteration in cell shape and surface morphology.

Effects of Interleukin-1 and Lipopolysaccharides on Protein and Carbohydrate Metabolism in Bovine Articular Cartilage Organ Cultures

The long term (18 day) metabolic response of bovine articular cartilage to treatment with either E. Coli lipopolysaccharide (LPS) or interleukin 1 was studied. For LPS treatment, incorporation of [35S]sulfate into the large proteoglycan population was inhibited 80% while that into the small interstitial proteoglycans was only inhibited 40%. Incorporation of [3H]serine into the large proteoglycan population was inhibited approximately 72% while incorporation into other protein was inhibited only 16%. Furthermore, the rate of catabolism of [3H]serine labeled proteoglycans was increased 2-fold by LPS treatment while the rate of 3H-labeled general protein catabolism was not affected. Incorporation of [3H]glucosamine into hyaluronate was increased; however a correction for changes in the specific activity of the intracellular [3H]glucosamine precursor pool in LPS-treated cultures indicated that the net amount of hyaluronate synthesized was not altered by LPS treatment. The 3H/35S ratios in isolated chondroitin sulfate disaccharides labeled with [35S]sulfate and [3H]glucosamine precursors were significantly changed during long term LPS treatment, suggesting that general carbohydrate pathways are altered. The 3H/35S changes were larger in the disaccharides isolated from the small proteoglycans indicating that different precursor pools, probably in different cell populations, preferentially synthesize this proteoglycan population. Interleukin-1 affected the same chondrocytic pathways as LPS as shown by a) the extent of inhibition of proteoglycan synthesis, b) the selective inhibition of synthesis of the large proteoglycan species, c) acceleration of proteoglycan catabolism, d) net depletion of proteoglycans from the tissue, e) increases in guanidine HCl extractable [3H]hyaluronate, f) increases in levels of prostaglandin E2 synthesis, g) changes in 3H/35S ratios in glycosaminoglycan chains and, h) minimal effects on general protein synthesis.

Monoclonal antibodies directed against epitopes within the core protein structure of the large aggregating proteoglycan (aggrecan) from the swarm rat chondrosarcoma.

The core protein of the large hyaline cartilage proteoglycan, aggrecan, is composed of six distinct domains: globular 1 (G1), interglobular, globular 2 (G2), keratan sulfate attachment, chondroitin sulfate (CS) attachment, and globular 3 (G3). Monoclonal antibodies that recognize epitopes in these domains were raised against Swarm rat chondrosarcoma aggrecan that was either denatured through reduction and alkylation or partially deglycosylated through chondroitinase ABC digestion or alkali elimination, the latter with or without sulfite addition. Monoclonal antibodies were further characterized for reactivity to purified aggrecan substructures including rat chondrosarcoma G1 and CS attachment domains, a recombinant rat chondrosarcoma G3 domain fusion protein, bovine articular cartilage G2 domain, and rat chondrosarcoma link protein (LP). Biochemical characterization of the specificities of these monoclonal antibodies indicated that one (1C6) recognized an epitope shared by both the G1 and the G2 domains; one (5C4) recognized an epitope shared by both LP and the G1 domain; one (7D1) recognized an epitope shared by both the G1 and the CS attachment domains; two (14A1 and 15B2) recognized epitopes in the CS attachment domain; one (14B4) recognized an epitope in the G3 domain; and one (13D1) recognized a ubiquitous epitope shared by the G1, G2, G3, and CS attachment domains of aggrecan and also LP. Collectively the specificities of these antibodies confirm the occurrence of multiple repeated epitopes (both carbohydrate and protein in nature) throughout the different domain structures of aggrecan. These antibodies have been proven to be useful for identifying aggrecan-like molecules in several connective tissues other than cartilage.