scholarly journals Glomerular mesangial cells in vitro synthesize an aggregating proteoglycan immunologically related to versican

1994 ◽  
Vol 302 (1) ◽  
pp. 49-56 ◽  
Author(s):  
G J Thomas ◽  
M T Bayliss ◽  
K Harper ◽  
R M Mason ◽  
M Davies
Keyword(s):  
Mesangial Cell ◽  
Mesangial Cells ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Gel Chromatography ◽  
Glomerular Mesangial Cells ◽  
Glomerular Cell ◽  
Core Proteins

Recent studies have shown that mesangial cells derived from human adult glomeruli synthesize a number of 35S-labelled proteoglycans including a large chondroitin sulphate proteoglycan (CSPG), two dermatan sulphate proteoglycans (biglycan and decorin) and two heparan sulphate proteoglycans [Thomas, Mason and Davies (1991) Biochem. J. 277, 81-88]. In the present study we have examined the interaction of these proteoglycans with hyaluronan (HA) using associative gel chromatography. Only the large CSPG bound to HA, with 60% of those molecules in the medium and 80% of those in the cell layer being able to interact. Reduction and alkylation, or treatment of the monomer CSPG with proteinases, prevented the formation of aggregates, suggesting that the core protein was involved. The aggregates formed between purified CSPG and HA could be dissociated in the presence of HA-oligosaccharides of at least 10 monosaccharides in length. The inclusion of link protein with CSPG and HA promoted the formation of aggregates. Experiments with 3H-labelled mesangial-cell proteoglycans confirmed that only the large CSPG, with core protein molecular masses of 400 kDa and 500 kDa, interacted with HA. After chondroitin ABC lyase treatment of CSPG isolated from conditioned culture medium, several bands similar to those observed with 3H-labelled core proteins were identified using a polyclonal antiserum that recognizes versican. A monoclonal antibody recognizing the 1-C-6 epitope in the G1 and G2 globular regions of aggrecan did not recognize either mesangial-cell CSPG or bovine aortic versican. Northern-blot analysis confirmed that human mesangial cells express versican. Thus human mesangial large CSPG is a member of the versican family of proteoglycans. The interaction of CSPG and HA within the glomerulus may be important in glomerular cell migration and proliferation.

scholarly journals Mesangial cell proteoglycans: synthesis and metabolism.

1992 ◽  
Vol 2 (10) ◽  
pp. S88
Author(s):  
M Davies ◽  
G J Thomas ◽  
L D Shewring ◽  
R M Mason
Keyword(s):  
Culture Medium ◽  
Mesangial Cell ◽  
Dermatan Sulfate ◽  
Mesangial Cells ◽  
Core Protein ◽  
Glomerular Mesangial Cells ◽  
Cellular Processes ◽  
Specialized Function ◽  
In Cells

In cultures of human adult glomerular mesangial cells, large chondroitin sulfate proteoglycans (CSPG) and small dermatan sulfate proteoglycans (DSPG) are synthesized. The large CSPG has a core protein, M(r) of 400,000 (major) and M(r) of 500,000 (minor), and binds to hyaluronic acid to form large aggregates. The two small DSPGs (Mr of approximately 350,000 and M(r) of approximately 200,000) were related to biglycan and decorin, respectively. The majority of these proteoglycans were located in the culture medium, but a hydrophobic form of the CSPG was extracted from the cell layer. Mesangial cells in the growing phase synthesized and secreted all three types of proteoglycans, but in cells arrested in G0 by serum deprivation the incorporation of (35S)sulfate in CSPG was drastically reduced. In the same cells stimulated to proliferate by replacing the medium with one containing serum, the synthesis of CSPG dramatically enhanced. The synthesis of CSPG and DSPG was also elevated in cells cocultured with cytokines but in contrast was significantly reduced when cultured in medium containing hyperglycemic levels of glucose. Finally, preliminary experiments are reported that indicate that CSPG and DSPG bind to low-density lipoproteins in vitro. These observations suggest a possible specialized function for proteoglycans in cellular processes characteristic of glomerular disease.

scholarly journals Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts

1985 ◽  
Vol 232 (1) ◽  
pp. 161-168 ◽  
Author(s):  
S Johansson ◽  
K Hedman ◽  
L Kjellén ◽  
J Christner ◽  
A Vaheri ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Human Articular Cartilage ◽  
Sulphate Proteoglycan ◽  
Fibroblast Cultures ◽  
Core Proteins ◽  
The Matrix

Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called ‘link proteins’ were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix.

scholarly journals β-d-xylosides and their analogues as artificial initiators of glycosaminoglycan chain synthesis. Aglycone-related variation in their effectiveness in vitro and in ovo

1987 ◽  
Vol 241 (2) ◽  
pp. 591-601 ◽  
Author(s):  
M Sobue ◽  
H Habuchi ◽  
K Ito ◽  
H Yonekura ◽  
K Oguri ◽  
...  
Keyword(s):  
Growth Rate ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Average Length ◽  
Heparan Sulphate ◽  
Carbon Number ◽  
Average Chain Length ◽  
In Ovo ◽  
Ability To Act

A series of aryl and alkyl O-beta-D-xylosides and their analogues with S, NH or CH2 in the glycosidic linkage were prepared and examined for their ability to act as artificial chain initiators of chondroitin (dermatan) sulphate synthesis in embryonic chick cartilage, foetal rat skin and 6-week-old-rat aorta under conditions where normal protein-core synthesis was inhibited by cycloheximide. For all these tissues in culture, phenyl O-beta-D-xyloside and phenyl beta-D-thioxyloside were clearly more effective than the corresponding N-xyloside and homo-C-xyloside. Introduction of a carboxy group to the para position of their aglycone yielded derivatives with far lower initiator activity. In a concentration range lower than 0.1 mM, the effectiveness of alkyl beta-D-thioxyloside was greatly influenced by the carbon number (n) of the alkyl group and was at a maximum at n = 7 or 8 for the cartilage, at n = 5 for the skin and at n = 4 for the aorta. In the beta-xyloside-treated cartilages, the average length of newly formed chondroitin sulphate chains reflected the chain-initiator activity of added xyloside, i.e. the higher the initiator activity, the shorter the average chain length. In the skin and aorta, none of the drugs could relieve the inhibition of heparan sulphate synthesis caused by cycloheximide. Fertilized hens' eggs were each injected on day 9 with 9.2 mumol of beta-xyloside and the skeletal systems of embryos were examined a week later. The embryos treated with beta-xylosides of relatively high initiator activity showed a 30-40% decrease in the overall growth rate of skeletons, whereas those treated with beta-xylosides of low initiator activity showed little or no decrease in the growth rate. The results are consistent with the notion that the observed change in skeletal morphology results mainly, if not completely, from beta-xyloside-induced synthesis of core-protein-free chondroitin sulphate, and further suggest that a procedure employing a series of beta-xyloside homologues with various initiator activities will furnish an easily applied criterion on which to test the specificity of xyloside action on biological processes.

Hormonal modulation of glomerular function

1983 ◽  
Vol 244 (2) ◽  
pp. F95-F104 ◽  
Author(s):  
L. D. Dworkin ◽  
I. Ichikawa ◽  
B. M. Brenner
Keyword(s):  
Plasma Flow ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Renal Plasma Flow ◽  
Hydraulic Pressure ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Glomerular Function ◽  
Hormonal Modulation

Glomeruli contain receptors for many hormones. Binding of angiotensin II (ANG II) or antidiuretic hormone (ADH) to glomerular mesangial cells elicits a contractile response. Other hormones induce synthesis of cyclic nucleotides (cAMP, cGMP). Glomeruli also synthesize several prostaglandins, renin, and ANG II. Micropuncture studies in Munich-Wistar rats have examined the effects of vasoactive drugs and hormones on the filtration process. Several vasodilators increase renal plasma flow in the dog and rat, but GFR remains relatively unchanged due to an offsetting fall in the ultrafiltration coefficient (Kf). Vasoconstrictor substances such as ANG II and norepinephrine cause declines in renal plasma flow and Kf, but GFR remains constant due to an increase in the transcapillary hydraulic pressure gradient. Antidiuretic peptides and parathyroid hormone also reduce Kf. Glomerular mesangial cells may regulate Kf by contracting and reducing glomerular capillary surface area. ANG II and ADH directly stimulate mesangial cell contraction in vitro. Other hormones appear to cause contraction by inducing local ANG II synthesis. These hormonal pathways are implicated in the pathogenesis of altered glomerular function in diverse forms of renal injury.

scholarly journals Characterization of proteoglycans isolated from associative extracts of human articular cartilage

1993 ◽  
Vol 293 (1) ◽  
pp. 165-172 ◽  
Author(s):  
V Vilím ◽  
A J Fosang
Keyword(s):  
Articular Cartilage ◽  
Molecular Mass ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Gel Chromatography ◽  
Human Articular Cartilage ◽  
Keratan Sulphate ◽  
Core Proteins ◽  
Sds Page

Approx. 10% of the total proteoglycan content of normal young human articular cartilage was extracted under associative conditions with Dulbecco's PBS. Proteoglycans isolated from the extract by Q-Sepharose chromatography were separated by gel chromatography and characterized by gradient gel SDS/PAGE and immunoblotting. Three species of small proteoglycans, two main populations of aggrecan and a population of its smaller fragments were identified. The major populations of aggrecan contained chondroitin sulphate chains, all or part of the N-terminal G1 and G2 domains and, therefore, intact keratan sulphate domains. The larger population was estimated by gradient SDS/PAGE to have a molecular mass of approx. 600 kDa or greater. The second population had an apparent molecular mass of approx. 300-600 kDa. Core proteins derived from these populations of proteoglycans separated on SDS/PAGE into several clusters of bands in the range from 120 to approx. 360 kDa. The extract further contained smaller fragments which lacked chondroitin sulphate but reacted with antibodies against keratan sulphate, and against epitopes present in the G2 domain of aggrecan. The presence of the G2 domain in a broad range of populations of decreasing size indicated extensive cleavage of the aggrecan core protein within its chondroitin sulphate domain. These findings suggest that fragmentation of aggrecan probably occurs in vivo in normal articular cartilage of young individuals. Associative extracts also contained decorin, biglycan and fibromodulin. These were resolved from aggrecan by gel chromatography and identified by immunodetection.

scholarly journals Characterization of proteoglycans synthesized by human adult glomerular mesangial cells in culture

1991 ◽  
Vol 277 (1) ◽  
pp. 81-88 ◽  
Author(s):  
G J Thomas ◽  
R M Mason ◽  
M Davies
Keyword(s):  
Culture Medium ◽  
Mesangial Cells ◽  
Cell Layer ◽  
Heparan Sulphate ◽  
Glomerular Mesangial Cells ◽  
Heparan Sulphate Proteoglycan ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Human Adult ◽  
Core Proteins

1. The newly synthesized proteoglycans from human adult glomerular mesangial cells labelled in vitro for 24 h with [35S]sulphate have been characterized using biochemical and immunological techniques. 2. The following proteoglycans were identified (% of total synthesized). (i) A large chondroitin sulphate proteoglycan, CSPG-I, Mr approximately 1 x 10(6) (10.6%). This proteoglycan consisted of a protein core of Mr approximately 4 x 10(5) and glycosaminoglycan chains of Mr 2.5 x 10(4), and was present in both the cell layer and the culture medium. (ii) A major small dermatan sulphate proteoglycan, DSPG-I, Mr 3.5 x 10(5) (46%), which was mainly located in the culture medium. (iii) A second minor small dermatan sulphate, DSPG-II, Mr approximately 2 x 10(5) (9.8%). This molecule was exclusively located in the culture medium. (iv) A large heparan sulphate proteoglycan, HSPG-I, Mr 8 x 10(5) (3.3%). (v) A second large heparan sulphate proteoglycan HSPG-II, Mr approximately 6 x 10(5) (23%). HSPG-I and HSPG-II were extracted from both the culture medium and the cell layer. 3. Western blot analysis of the core proteins released by chondroitin ABC lyase treatment of DSPG-I and DSPG-II identified these dermatan sulphate proteoglycans as biglycan and decorin respectively. Both DSPG-I and DSPG-II had core proteins of Mr 45,000. 4. The cell-layer-associated forms of CSPG-I, HSPG-I and HSPG-II were accessible to limited trypsin treatment, bound to octyl-Sepharose and could be inserted into liposomes, indicating a possible cell membrane location. 5. Pulse-chase experiments indicated that the cell-layer-associated [35S]proteoglycans undergo limited metabolism to inorganic [35S]sulphate, the majority of which is accounted for by the degradation of HSPG-II and to a lesser extent DSPG-I.

A mechanism by which atrial natriuretic factor mediates its glomerular actions

1986 ◽  
Vol 251 (6) ◽  
pp. F1036-F1042 ◽  
Author(s):  
R. G. Appel ◽  
J. Wang ◽  
M. S. Simonson ◽  
M. J. Dunn
Keyword(s):  
Mesangial Cell ◽  
Mesangial Cells ◽  
Dependent Manner ◽  
Ang Ii ◽  
Glomerular Mesangial Cells ◽  
Sprague Dawley ◽  
Cell Contraction ◽  
Concentration Dependent Manner ◽  
Cell Contractility

Differential in vivo glomerular effects of atriopeptin I (AP I) and atriopeptin III (AP III) were studied in parallel with in vitro physiological and biochemical parameters. In anesthetized Sprague-Dawley rats, AP III, but not AP I, significantly increased glomerular filtration rate. Image analysis microscopy was used to assess the effect of AP I and AP III on angiotensin II (ANG II)-induced contraction of cultured rat glomerular mesangial cells. AP III, but not AP I, inhibited ANG II-induced mesangial cell contraction in a concentration-dependent manner. Additional inhibitory agents included exogenous DBcGMP, 8-BrcGMP, Na nitroprusside, and DBcAMP. AP III stimulated mesangial cell cGMP with a lower threshold and greater maximum stimulation than AP I. Neither agent stimulated cAMP accumulation. Since mesangial cell contractility may regulate the glomerular capillary surface area, these results suggest that AP III partially mediates its glomerular effects through inhibition of ANG II-induced mesangial cell contraction. Whereas cGMP is not clearly implicated as the mediator of this effect, it appears that both cGMP and cAMP may regulate the state of mesangial cell contractility.

scholarly journals Endothelium-derived relaxing factor, nitric oxide: effects on and production by mesangial cells and the glomerulus.

1993 ◽  
Vol 3 (8) ◽  
pp. 1435-1441
Author(s):  
L Raij ◽  
P J Shultz
Keyword(s):  
Nitric Oxide ◽  
Vascular Tone ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Glomerular Mesangial Cells ◽  
Relaxing Factor ◽  
Endothelium Derived Relaxing Factor

The endothelium-derived relaxing factor nitric oxide (EDRF/NO) is a labile, endogenous vasodilator that is important in the control of systemic vascular tone. This review focuses on the effects of EDRF/NO on glomerular mesangial cells in vitro and on the role of EDRF/NO in mesangial and glomerular physiology and pathophysiology in vivo. It was concluded that EDRF/NO can stimulate increases in cGMP, inhibit mesangial cell contraction, and inhibit growth factor-induced proliferation of mesangial cells in culture. Furthermore, incubation with endotoxin or cytokines stimulates mesangial cells to produce EDRF/NO, via an inducible NO synthase enzyme. Therefore, it is likely that NO could play a role in the inflammatory response within the glomerulus. Finally, recent studies providing evidence that EDRF/NO is functional within the glomerulus in vivo, especially during endotoxemia and inflammation are also reviewed.

scholarly journals Proteoglycan metabolism in the connective tissue of pregnant and non-pregnant human cervix. An in vitro study

1991 ◽  
Vol 275 (2) ◽  
pp. 515-520 ◽  
Author(s):  
M Norman ◽  
G Ekman ◽  
U Ulmsten ◽  
K Barchan ◽  
A Malmström
Keyword(s):  
Connective Tissue ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
In Vitro Study ◽  
Gel Chromatography ◽  
Cervical Tissue ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycans

Profound changes occur in the cervix during pregnancy. In particular, the connective tissue is remodelled. To elucidate the mechanisms behind this process, the metabolism of cervical connective tissue was studied using tissue cultures. Cervical biopsies from non-pregnant and pregnant women were incubated with [35S]sulphate. The proteoglycans of the tissue specimens were purified by ion-exchange and gel chromatography and characterized by SDS/PAGE and by enzymic degradation. In the non-pregnant cervix, the incorporation of [35S]sulphate into the proteoglycans was linear for 48 h. During the first 6 h of incubation the accumulation of chiefly one small labelled proteoglycan (apparent Mr 110,000) substituted with dermatan sulphate was recorded. This is in accordance with the known proteoglycan composition of non-pregnant cervical tissue. In addition, small amounts of two larger radioactive dermatan/chondroitin sulphate proteoglycans (apparent Mr values 220,000 and greater than 500,000) were recorded. After longer periods of incubation the proportion of heparan sulphate proteoglycans increased considerably. The pregnant tissue showed a clearly different composition of labelled proteoglycans. An increased accumulation of the two larger dermatan/chondroitin sulphate proteoglycans was seen in addition to the dominant small dermatan sulphate proteoglycan of the non-pregnant cervix. The rate of accumulation of these two proteoglycans was about 3 times higher in the pregnant tissue, whereas that of the small dermatan sulphate proteoglycan was only increased 2-fold. The fact that the concentration of proteoglycans in the pregnant cervix is approximately one-half of that in the non-pregnant cervix indicates that the turnover of proteoglycans in pregnant cervical tissue is significantly increased. The major effect of this profound change of metabolism was a 50% decrease in proteoglycan content and a 2-fold increased proportion of a dermatan sulphate proteoglycan with an apparent Mr of 220,000.

scholarly journals Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone

1987 ◽  
Vol 248 (3) ◽  
pp. 801-805 ◽  
Author(s):  
A A Day ◽  
C I McQuillan ◽  
J D Termine ◽  
M R Young
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Homologous Sequence ◽  
Bovine Bone ◽  
Amino Acid Homology ◽  
Core Proteins ◽  
Attachment Sites

The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.

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