Multi-membrane chitosan hydrogels as chondrocytic cell bioreactors.

We investigated the bioactivity of new chitosan-based multi-membrane hydrogel (MMH) architectures towards chondrocyte-like cells. The microstructure of the hydrogels constituting the membranes precludes any living cell penetration, whereas their lower scale architecture allows the protein diffusion. The biological behavior of chondrocytes implanted within the MMH inter-membrane spaces was studied for 45 days in culture. Chondrocytes formed cell aggregates and proliferated without loosing their chondrogenic phenotype as illustrated by collagen II and aggrecan expressions at the mRNA and protein levels. Cells produced neo-formed alcyan blue matrix proteins filling MMH interspaces. The HiF-2α/SOX9 pattern of expression suggested that the elevated chondrocytic phenotype in MMH could be related to a better hypoxic local environment than in classical culture conditions. Pro-inflammatory markers were not expressed during the period of culture. The low level of nitric oxide accumulation within the inter-membrane spaces and in the incubation medium implied that chitosan consumed nitrites produced by entrapped chondrocytes, in relation with the decrease of its molecular weight of 50%. Our data suggest that MMH structures may be considered as complex chondrocytic cell bioreactors; "active decoys of biological media", potentially promising for various biomedical applications like the inter-vertebral disk replacement.

Natural chondroitin sulphates increase aggregation of proteoglycan complexes and decrease ADAMTS-5 expression in interleukin 1 beta-treated chondrocytes.

OBJECTIVE: To assess the effect of natural chondroitin sulphate (CS) on the ability of neosynthesized sulphated proteoglycans (PGs) to aggregate in cultured chondrocytes treated with interleukin (IL)1 beta. METHODS: Primary cultured rabbit articular chondrocytes were treated or not with IL1 beta alone or with concentrations of CS for 20 h. Neosynthesized PGs were labelled by incorporation of [35SO(4)]-sulphate and analysed by chromatography on Sepharose 2B columns. Gelatinolytic activity was measured by zymography, and matrix metalloproteinase (MMP)1 mRNA level in chondrocytes underwent real-time PCR. Expression of ADAMTS (for "a disintegrin and metalloproteinase with thrombospondin motifs") -4 and -5 was analysed by real-time PCR and western blotting. RESULTS: The production of [35SO(4)]-labelled PGs was significantly increased with 10 microg/ml CS in the cellular pool rather than in the incubation medium. The addition of CS to IL1 beta-treated cells inhibited in part the disaggregation of sulphated PGs induced by IL1 beta. This inhibitory effect of CS is associated with a significant decrease in ADAMTS-5 expression at the mRNA and protein levels. No effect of CS was observed on IL1 beta-induced gelatinolytic activity, MMP1 mRNA expression or ADAMTS-4 expression. CONCLUSION: CS increases the production of functional sulphated PGs in the direct environment of chondrocytes in vitro. This beneficial effect of CS in IL1 beta-treated cells is associated with decreased expression of ADAMTS-5.

A material decoy of biological media based on chitosan physical hydrogels: application to cartilage tissue engineering.

The cartilage tissue has a limited self-regenerative capacity. Tissue-engineering represents a promising trend for cartilage repair. The present study was aimed to develop a biomaterial formulation by combining fragments of chitosan hydrogel with isolated rabbit or human chondrocytes. We first reported the properties of the constructs elaborated with rabbit chondrocytes and pure chitosan physical hydrogels with defined molecular weight, acetylation degree and polymer concentration. Morphological data showed that chondrocytes were not penetrating the hydrogels but tightly bound to the surface of the fragments and spontaneously formed aggregates of combined cell/chitosan. A significant amount of neo-formed cartilage-like extracellular matrix (ECM) was first accumulated in-between cells and hydrogel fragments and furthermore was widely distributed within the neo-construct. The optimal biological response was obtained with hydrogel fragments concentrated at 1.5% (w/w) of polymer made from a chitosan with a degree of acetylation between 30 and 40%. Such hydrogels were then mixed with human chondrocytes. The phenotype of the cells was analyzed by using chondrocytic (mRNA expression of mature type II collagen and aggrecan as well as secretion of proteoglycans of high molecular weight) and non chondrocytic (mRNA expression of immature type II collagen and type I collagen) molecular markers. As compared with human chondrocytes cultured without chitosan hydrogel which rapidly dedifferentiated in primary culture, cells mixed with chitosan rapidly loose the expression of type I and immature type II collagen while they expressed mature type II collagen and aggrecan. In these conditions, chondrocytes maintained their phenotype for as long as 45 days, thus forming cartilage-like nodules. Taken together, these data suggest that a chitosan hydrogel does not work as a scaffold, but could be considered as a decoy of cartilage ECM components, thus favoring the binding of chondrocytes to chitosan. Such a biological response could be described by the concept of reverse encapsulation.

Peroxisome proliferator-activated receptor gamma and its ligands in controlling interleukin-1beta target gene expression: a confusing story.

Antiinflammatory effects by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have been previously reported. However, PPARgamma dependency and the molecular mechanism involved in these effects require more investigation to clearly demonstrate whether PPARgamma is a key modulator of the antiinflammatory process. This would permit the design of more specific agonists or antagonists able to address the gamma subtype without cross reactions with other transcription factors, thus preventing undesirable side effects. However, several hurdles need to be taken into consideration, such as the coexpression of several PPAR isotypes in the same cell type. As PPARgamma and -alpha seem to play equal antiinflammatory roles, determining the subset of specific PPAR subtype target genes appears to be crucial. The work described here is our current understanding of the modulations of interleukin-1 target gene expression by PPARgamma and its ligands.

Dual effects of 17beta-oestradiol on interleukin 1beta-induced proteoglycan degradation in chondrocytes.

OBJECTIVE: To determine whether 17beta-oestradiol (E2) modulates interleukin (IL) 1beta-induced proteoglycan degradation in chondrocytes, and to analyse the part played by metalloproteinases (MMPs) in this process. METHODS: Primary cultured rabbit articular chondrocytes were prepared and treated with 10 ng/ml IL1beta combined or not with 0.1-10 nM E2. Neosynthesised proteoglycans (PGs) were evaluated after incorporation of [(35)SO(4)]sulphate and further analysed after chromatography on a Sepharose 2B column. Chondrocyte mRNA levels of aggrecan, MMP-1, -3, -13, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were studied by northern blot. MMP-1 activity was measured by zymography. MMP-1 gene transcription was studied by transient transfection of chondrocytes with an MMP-1-luciferase construct. RESULTS: E2 modulated the IL1beta-induced total sulphated PGs in rabbit articular chondrocytes, which decreased as the E2 concentration was increased. At a low concentration (0.1 nmol/l) E2 counteracts the IL1beta-induced decrease in sulphated PG, while at high concentration (10 nmol/l) E2 enhances the IL1beta effects. A biphasic E2 effect was also observed on IL1beta-induced disaggregation of PG, 53-58 kDa gelatinolytic activity, and MMP-1, -3, and -13 mRNA levels. In contrast, E2 did not modify the level of aggrecan mRNA and had no effect on TIMP-1 mRNA expression. Finally, simultaneous addition of IL1beta and E2 (0.1-10 nmol/l) did not modify IL1beta-induced MMP-1-luciferase activity, suggesting that E2 effects probably occur at the post-transcriptional level of MMP gene expression. CONCLUSION: Oestrogen concentration may have an inverse effect on IL1beta stimulated proteoglycan degradation and MMP production by chondrocytes.

[Cell therapy and its clinical applications. Cartilage cell therapy, present and future]. / La thérapie cellulaire dans ses applications cliniques. Thérapie cellulaire du cartilage, présent et futur.

Articular cartilage has a very poor capacity for repair. In order to get a normal functional efficacy, the replaced tissue has to reproduce the structure, composition and physico-chemical properties of native cartilage tissue. The transplantation of cultured autologous chondrocytes into chondral defects is currently applicable only in the case of young sportive people with a limited lesion in an otherwise relatively normal joint. Recent experimental studies have shown that pluripotent mesenchymal cells from bone marrow could also repair experimental osteochondral defects. An advantage of this grafting procedure is that large areas of cartilage surface could be covered. Bone marrow cells are not so difficult to get, they have a high potency to divide and they can develop in vitro as chondrogenic, osteogenic or adipogenic cells. The present ways of research are: to characterize one or several growth factors capable to specifically induce the chondrogenic lineage; to determine nutrient and environmental conditions allowing the cultured chondrogenic cells to undergo a maturation process within the cell pellet; to elaborate three-dimensional synthetic, biodegradable polymeric scaffolds assessed with respect to chondrogenic cell adhesion, proliferation, maturation and cartilage matrix secretion; finally, to elaborate a mixed biomaterial composed of chondrogenic and osteogenic cells selectively distributed within polymeric scaffolds in order to get a better adherence of the implanted cells to the lesion sites.

Disk degeneration and disk herniation: the contribution of mechanical stress.

Experimental studies on the role for mechanical stresses in the genesis of disk degeneration and herniation are reviewed. Simple mechanical stimulations of functional vertebral segments cannot cause a disk herniation: a complex mechanical stimulation combining forward and lateral bending of the spine followed by violent compression is needed to produce posterior herniation of the disk. Intervertebral disk degeneration seems to influence the development of posterior disk herniation or foraminal disk protrusion. Furthermore, direct mechanical stimulation of the disk tissue or cells generates complex metabolic and cellular responses that lead to qualitative and quantitative modulation of disk matrix proteins. Thus, it is becoming increasingly likely that physical and metabolic factors act in concert to produce disk herniation.

Monolayer anulus fibrosus cell cultures in a mechanically active environment: local culture condition adaptations and cell phenotype study.

Intervertebral disc cells can be cultured in vitro. Several culturing systems in a mechanically active environment have been developed to study the relationship between mechanical stimulations and biochemical events. The aim of this study was to assess the phenotype of rabbit intervertebral disc cells from the anulus fibrosus (AF) region cultured on flexible substrate before and after application of cyclic tensile stretch (CTS) and to control culture conditions during application of CTS. CTS was applied with a pressure-operated instrument, inducing the deformation of flexible-bottomed culture plates (Flexercell) at 20% and 5% stretch, at a frequency of 1 Hz, during 30 minutes to 24 hours. A significant decrease in culture medium volume and temperature was observed (52% and 2.1 degrees C at 20% stretch and 24 hours' application of CTS). These phenomena were inhibited by adding culture medium around culture wells and by a culture medium temperature control system. Like AF cells cultured in plastic wells, AF cells cultured on flexible substrate expressed collagen type II, but collagen type I mRNA was not detected. In both culture conditions, neosynthesized proteoglycans had the same aggregating properties. CTS at 20% stretch during 12 hours did not induce cell detachment from the substrate and did not modify aggregating properties of neosynthesized proteoglycans; AF cells continued to express collagen type II but not collagen type I mRNA. In conclusion, the Flexercell system appears to be appropriate for studying, at the cellular level, the metabolic responses to CTS.

Sensitivity of anulus fibrosus cells to interleukin 1 beta. Comparison with articular chondrocytes.

STUDY DESIGN: Anulus fibrosus cells from rabbits were grown in primary culture 1) to study their ability to produce prostaglandin E2 and Type II phospholipase A2, and to express stromelysin-1 messenger ribonucleic acid; and 2) to study the effect of interleukin 1 beta on this production and on proteoglycan aggregation. OBJECTIVES: To investigate the potency of anulus fibrosus cells to respond to interleukin 1 beta by producing degradative and inflammatory agents as compared with the potency of articular chondrocytes in the same animal. SUMMARY OF BACKGROUND DATA: Interleukin 1 beta has been implicated in the degradation of intervertebral discs. The way anulus fibrosus cells differ from articular chondrocytes in their responses to interleukin 1 beta remains to be established. METHODS: Anulus fibrosus cells and articular chondrocytes were obtained from young rabbits, grown in primary culture, and incubated with interleukin 1 beta. The newly synthesized proteoglycan was measured by labeling with [35S]-sulfate. Proteoglycan aggregation was analyzed by the elution profile on Sepharose 2B columns. The contents of collagen Type II and stromelysin-1 messenger ribonucleic acid were assessed by Northern blot analysis. The Type II phospholipase A2 activity was measured using a fluorometric substrate. Prostaglandin E2 production was evaluated by radioimmunoassay. RESULTS: Anulus fibrosus cells had 2.5-fold less Type II collagen messenger ribonucleic acid than articular chondrocytes, and interleukin 1 beta had no significant effect on this. Anulus fibrosus cells synthesized and secreted four-fold less proteoglycan than articular chondrocytes. Interleukin 1 beta reduced the anulus fibrosus content of total [35S]-sulfated proteoglycan by 35% (P < 0.01), and that of articular cells by 41% and decreased proteoglycan aggregation. Interleukin 1 beta induced the production of stromelysin-1 messenger ribonucleic acid in both cell types. The stromelysin-1 messenger ribonucleic acid content of anulus fibrosus cells was one half that of articular cells. Interleukin 1 beta increased the production of prostaglandin E2 and caused a dose-dependent secretion of Type II phospholipase A2 activity in both cell types. Its effect was 2.5-fold lower in anulus fibrosus cells than in articular chondrocytes. CONCLUSION: Anulus fibrosus cells can be stimulated by interleukin 1 beta to produce factors implicated in local degradative and inflammatory processes. This production is associated with decreased proteoglycan aggregation. Anulus fibrosus cells respond slightly less well to interleukin 1 beta in vitro than do articular cells.

Phenotypic characteristics of rabbit intervertebral disc cells. Comparison with cartilage cells from the same animals.

STUDY DESIGN: Intervertebral disc cells were extracted from the surrounding matrix, and their metabolic activities and phenotypes were studied. OBJECTIVES: To compare the metabolic activities and phenotypes of cell populations extracted from the intervertebral discs of young rabbits with those of articular and growth plate chondrocytes from the same animals. SUMMARY OF BACKGROUND DATA: The phenotype of intervertebral disc cells has been poorly studied and still is debated. METHODS: The intervertebral discs as well as articular and vertebral growth plate cartilage of rabbits were digested enzymatically. The morphology of freshly isolated cells was examined. Their contents of collagen II and X mRNAs were determined by Northern blot analysis, and their sulfation activity by 35S-sulfate incorporation as chondrocytic markers. Cells were cultured at high density or low density and grown in primary culture. The stability of their phenotype was monitored by evaluating the collagen I and II mRNA ratio. The proteoglycans newly synthesized by the cells also were quantified, and their elution profile analyzed on Sepharose 2B columns. RESULTS: The anulus fibrosus cells were morphologically undistinguishable from articular chondrocytes. The nucleus pulposus contained mainly large vacuolated cells and a few smaller cells. All freshly extracted cells expressed different levels of collagen II mRNA. Anulus fibrosus and nucleus pulposus cells contained, respectively, 22% and 8% of collagen II mRNA compared with that found in articular or growth plate chondrocytes from the same animal. Only growth plate chondrocytes expressed collagen X. When anulus fibrosus cells were incubated for 48 hours at high density, they had collagen II mRNA contents similar to those of articular and growth plate chondrocytes, but synthesized five to six times fewer sulfated proteoglycans. When seeded at low density, anulus fibrosus cells divided more slowly than articular chondrocytes and incorporated four times fewer 35S-sulfate into proteoglycans. Their collagen II mRNA content was 2.75-fold lower than that of chondrocytes, and the procollagen alpha 1II/alpha 1I mRNA ratio was 3.1 for anulus fibrosus cells and 7 for chondrocytes. No collagen X mRNA was detected. When incubated for 48 hours at high density, the nucleus pulposus giant cells had four times less collagen II mRNA content than cartilage cells but synthesized the same amounts of sulfated proteoglycans. They did not divide during 21 days in culture and still contained collagen II mRNA but no collagen X mRNA. CONCLUSIONS: Findings showed that intervertebral disc cells all express cartilage-specific matrix proteins with quantitative differences, depending on their anatomic situation. It is suggested that anulus fibrosus cells are chondrocytic cells at a different stage of differentiation than articular and growth plate chondrocytes. The phenotype of nucleus pulposus cells still is unclear. They could be chondrocytic or notochordal. A definitive answer to this important question requires differentiating markers of notochordal cells.

Posttranscriptional effect of insulin-like growth factor-I on interleukin-1beta-induced type II-secreted phospholipase A2 gene expression in rabbit articular chondrocytes.

Large amounts of type II-secreted phospholipase A2 (type II sPLA2) are secreted into inflammatory synovial fluid and they are believed to induce the synthesis of lipid mediators by articular chondrocytes. Preliminary experiments showed that insulin-like growth factor-I, which counteracts cartilage degradation in arthritis, inhibits interleukin-1beta-induced type II sPLA2 gene expression in rabbit articular chondrocytes (Berenbaum, F., G. Thomas, S. Poiraudeau, G. Bereziat, M.T. Corvol, and J. Masliah. 1994. FEBS Lett. 340: 51-55). The present study showed that IL-1beta induced the sustained synthesis of prostaglandin E2 and a parallel increase in type II sPLA2 gene expression (assessed by enzymatic activity and Northern blot analysis), but no increase in cytosolic PLA2 gene expression (assessed by Northern and Western blot analysis) or cytosolic PLA2 activity in rabbit articular chondrocytes. IGF-I inhibited both IL-1beta-stimulated PGE2 synthesis and type II sPLA2 gene expression, but had no effect on cytosolic PLA2 gene expression. Nuclear run-on experiments revealed that IL-1beta stimulated the transcription rate of type II sPLA2 gene, giving rise to long-lived mRNA in cells treated with actinomycin D. IGF-I did not affect transcription rate, suggesting that it acts as a post-transcriptional step. Sucrose density gradient analysis of the translation step showed no effect of IGF-I on the entry of type II sPLA2 mRNA into the polysomal pool or on its distribution into the various polysomal complexes, suggesting that IGF-I does not act on the translation of the mRNA. Lastly, IGF-I strongly decreased the half-life of IL-1beta-induced type II sPLA2 mRNA (from 92 to 12 h), suggesting that IGF-I destabilizes mRNA. These data demonstrate that IL-1beta stimulates the transcription rate of the type II sPLA2 gene and gives rise to a very stable mRNA. In contrast, IGF-I decreases the half-life of the type II sPLA2 message.

Different mechanisms are involved in intracellular calcium increase by insulin-like growth factors 1 and 2 in articular chondrocytes: voltage-gated calcium channels, and/or phospholipase C coupled to a pertussis-sensitive G-protein.

This study describes the mechanisms involved in the IGF-1 and IGF-2-induced increases in intracellular calcium concentration [Ca2+]i in cultured chondrocytes and the involvement of type 1 IGF receptors. It shows that IGF-1, IGF-2, and insulin increased the cytosolic free calcium concentration [Ca2+]i in a dose-dependent manner, with a plateau from 25 to 100 ng/ml for both IGF-1 and IGF-2 and from 1 to 2 micrograms/ml for insulin. The effect of IGF-1 was twice as great as the one of IGF-2, and the effect of insulin was 40% lower than IGF-1 effect. Two different mechanisms are involved in the intracellular [Ca2+]i increase. 1) IGF-1 and insulin but not IGF-2 involved a Ca2+ influx through voltage-gated calcium channels: pretreatment of the cells by EGTA and verapamil diminished the IGF-1 or insulin-induced [Ca2+]i but did not block the effect of IGF-2. 2) IGF-1, IGF-2, and insulin also induced a Ca2+ mobilization from the endoplasmic reticulum: phospholipase C (PLC) inhibitors, neomycin, or U-73122 partially blocked the intracellular [Ca2+]i increase induced by IGF-1 and insulin and totally inhibited the effect of IGF-2. This Ca2+ mobilization was pertussis toxin (PTX) dependent, suggesting an activation of a PLC coupled to a PTX-sensitive G-protein. Lastly, preincubation of the cells with IGF1 receptor antibodies diminished the IGF-1-induced Ca2+ spike and totally abolished the Ca2+ influx, but did not modify the effect of IGF-2. These results suggest that IGF-1 action on Ca2+ influx involves the IGF1 receptor, while part of IGF-1 and all of IGF-2 Ca2+ mobilization do not implicate this receptor.

Circulating insulin-like growth factor system changes in women with acute estrogen deficiency induced by GnRH agonist.

This prospective longitudinal study was undertaken to examine the short-term effects (6 months) of estrogen withdrawal on the circulating IGF system. A series of 40 patients suffering from endometriosis was studied before and after a 6-month treatment period with gonadotrophin releasing hormone (GnRH) agonist and calcium, with or without nasal salmon calcitonin. The plasma concentrations of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) were measured by radioimmunoassay and radioreceptor assay respectively. Plasma IGF binding proteins (IGFBPs) were quantified and characterized by ligand blot and immunoblot. In all patients, a secondary hypoestrogenism was observed, including a 4% decrease in lumbar bone mineral density (L-BMD). The plasma IGF-I and IGF-II concentrations increased after treatment (24%, p < 0.0005 and 40%, p < 0.004 respectively), with no significant difference between the treatment groups. There was a positive correlation between plasma IGF-I (but not IGF-II) changes and changes in urinary deoxypyridinoline (r = 0.32, p < 0.05), urinary C telopeptide of type 1 collagen (r = 0.33, p < 0.04) and total plasma alkaline phosphatases (r = 0.33, p < 0.04). No correlation was found between IGF-I and L-BMD changes, while there was a positive correlation between the changes in plasma IGF-II and L-BMD (r = 0.32, p < 0.05). Ligand blot analysis revealed a significant increase in IGF-II binding to a 29-31 kilodalton region where positive staining with specific antibodies to IGFBP-3 or IGFBP-1 was observed. In conclusion, IGF-I and IGF-II plasma concentrations are both increased following a short period of treatment with a GnRH agonist. The changes in individual IGF peptides are differently correlated with changes in markers of bone remodelling and L-BMD.

[Cartilage degradation and articular inflammation]. / Dégradation du cartilage et inflammation articulaire.

Osteoarthritis is mainly characterized by cartilage degradation as a result of series of pathological processus still not well understood. In recent years much interest has centered on the contribution of cytokines to cartilage degradation: characterization of cytokines extracted from normal and pathological articulation, site of production (synoviocytes, chondrocytes, osteoblasts) and their role on the production of proteases, protease inhibitors or other types of molecules implicated in osteoarthritis. Several types of cytokines such as IL 1 beta and TNF alpha, known as proinflammatory factors in rheumatoid arthritis, have been shown to be present in the synovial fluid from osteoarthritic patients. Recent experimental data allow us to better understand the cellular and molecular mechanisms of the cytokines respectively involved into rheumatoid arthritis and osteo-arthritis.

Synergistic effect of interleukin-1 beta and tumor necrosis factor alpha on PGE2 production by articular chondrocytes does not involve PLA2 stimulation.

This study investigates the ways in which two proinflammatory cytokines, tumor necrosis factor alpha (TNF) and interleukin-1 beta (IL1), cause increased production of prostaglandin E2 (PGE2) in rabbit articular chondrocytes (RAC). Rabbit articular chondrocytes in primary culture were incubated with IL1, TNF, or both. Arachidonic acid (AA) release, PGE2 production, and the activities of cytosolic phospholipase A2 (cPLA2), secreted phospholipase A2 (sPLA2), and cyclooxygenase (COX) were measured. The mRNA levels of cPLA2, sPLA2, and COX-2 were also measured by Northern blotting, using specific complementary DNA probes. Incubation of IL1-stimulated RAC with TNF further increased PGE2 production. This synergy did not involve PLA2 stimulation, as there were no increases in AA release, cPLA2 and sPLA2 activities, or mRNA. In contrast, TNF increased the effect of IL1 on COX-2 activity and mRNA level. These results show that TNF and IL1 act in synergy in PGE2 production in articular chondrocytes. As sPLA2 and cPLA2 do not seem to be involved, COX-2 appears to be the best target for a specific anti-inflammatory strategy against cartilage degradation.

Insulin-like growth factor I, a unique calcium-dependent stimulator of 1,25-dihydroxyvitamin D3 production. Studies in cultured mouse kidney cells.

Previous in vivo and in vitro studies suggest that insulin-like growth factor (IGF-I) could be a regulator of the renal production of 1,25-(OH)2D3. In the present work, the local effect of low nanomolar concentrations of IGF-I on the 25-OH-D3-1 alpha-hydroxylase activity and the mechanism of its action have been investigated. To do so, an in vitro model of mouse proximal tubular cells in primary culture has been developed. These cells bear specific high affinity IGF-I binding sites (apparent Kd = 1.95 +/- 0.46 nM) and express the ability to convert [3H]25-(OH)D3 into [3H]1,25-(OH)2D3 (Km = 139 +/- 15.7 nM). Human recombinant IGF-I (10-100 ng/ml) stimulated both sodium-dependent phosphate uptake and 1,25-(OH)2D3 synthesis by these cells, in a time- and dose-dependent manner. IGF-I did not alter the apparent Michaelis constant but increased the maximum velocity of the 25-OH-D3-1 alpha-hydroxylase activity. This effect required protein synthesis. It was not affected by calphostin or GF109203X, two protein kinase C inhibitors, and was not mimicked by phorbol 12-myristate 13-acetate. In contrast, it was blocked by verapamil, a calcium channel blocker. Calcium depletion of the medium blunted the IGF-I effect but not that of human 1-34 parathyroid hormone 5 x 10(-8) M. IGF-I thus appears to be the first example of a physiological calcium-dependent regulator of the renal metabolism of vitamin D.

Inhibition of chondrocyte cathepsin B and L activities by insulin-like growth factor-II (IGF-II) and its Ser29 variant in vitro: possible role of the mannose 6-phosphate/IGF-II receptor.

Lysosomal enzymes and IGF-II both bind to the mannose 6-phosphate (M6P)/IGF-II receptor. This receptor targets newly synthesized lysosomal enzymes to lysosomes. The functional meaning of IGF-II binding to this receptor is not well known. We have postulated that IGF-II, the Ser29 IGF-II variant (vIGF-II) and IGF-I on lysosomal cathepsin B and L activities from post-natal rabbit chondrocytes in vitro. This effect was compared with the ability of each peptide to stimulate chondrocyte-sulfated proteoglycan synthesis. The sulfating dose-response relationship of the IGF peptides corresponded to their relative binding affinities for the type I-IGF receptor (IGF-I > IGF-II > vIGF-II). The intracellular cathepsin B and L activities were inhibited in a time- and dose-dependent manner by IGF-II or vIGF-II. Maximal inhibition of cathepsin B and L activities (40 and 30% below controls, respectively) was found after an 8 h treatment with 100 ng/ml IGF-II or vIGF-II. By contrast, IGF-I up to 1 micrograms/ml or insulin up to 2 micrograms/ml had no inhibitory effect. The relative potency pattern corresponded to the binding profile of each ligand for the M6P/IGF-II receptor. A treatment of chondrocytes with IGF-I or insulin transiently increased the binding of radiolabelled IGF-II at the cell surface to approximately 120% of controls, whereas IGF-II or vIGF-II had no effect. Thus, it is unlikely that the inhibition of lysosomal enzyme activities by IGF-II peptides could result from a redistribution of M6P/IGF-II receptors from intracellular compartments to the plasma membrane. We hypothesize that internalized IGF-II peptides could occupy the intracellular M6P/IGF-II binding sites required for targeting of cathepsins B and L to lysosomes.

Purification and characterization of insulin-like growth factor II (IGF II) and an IGF II variant from human placenta.

In order to purify variant IGF II peptides from human placenta, we have developed a purification procedure combining heparin affinity chromatography and cation-exchange, reversed-phase and size-exclusion HPLC. Two peptides were purified, both having apparent M(r) values of ca. 7300 Da as evaluated by SDS-PAGE. N-Terminal sequencing revealed IGF II and an IGF II variant in which Ser29 was replaced by the tetrapeptide Arg-Leu-Pro-Gly. The final yield of variant IGF II was about eight-fold lower than that of IGF II. Both pure peptides were functionally active as they bound to type I and type II IGF receptors from ovine and human placental membranes, as determined by crosslinking experiments and displacement curve studies.