Human insulin A-chain peptide analog(s) with in vitro biological activity.

In a previous study, we showed that a synthetic human insulin 1-chain analog, named analog (3) was capable of mimicking in vitro effects of native insulin, including stimulation of cell proliferation, glucose uptake and glycogen synthesis. Here, we have synthesized three new analogs (6, 9, 12) of the human A-chain, bearing or not their N- or C-terminal residue, to determine the structural features which are responsible for their biological properties. In vitro experiments clearly demonstrated that the N-terminal part of the peptides is required for the biological activity of the molecules, suggesting its crucial role in the mechanism underlying the cellular effect. Our findings may help to better understand the mechanism of interaction between insulin and its receptor. In addition, the present data demonstrate that some mini-insulin derived from the A-chain can exert similar effects as native insulin. These small peptides may offer specific advantages over insulin in the definition of new strategies for diabetes treatment.

Chondroitin sulfate increases hyaluronan production by human synoviocytes through differential regulation of hyaluronan synthases: Role of p38 and Akt.

OBJECTIVE: To uncover the mechanism by which chondroitin sulfate (CS) enhances hyaluronan (HA) production by human osteoarthritic (OA) fibroblast-like synoviocytes (FLS). METHODS: The production of HA was investigated by exposing human OA FLS to CS in the presence or absence of interleukin-1beta (IL-1beta). HA levels were determined by enzyme-linked immunosorbent assay, and levels of messenger RNA (mRNA) for HA synthase 1 (HAS-1), HAS-2, and HAS-3 were determined by real-time polymerase chain reaction analysis. The effect of CS and IL-1beta on signaling pathways was assessed by Western blotting. Specific inhibitors were used to determine their effects on both HA production and HAS expression. The molecular size of HA was analyzed by high-pressure liquid chromatography. RESULTS: CS increased HA production by FLS through up-regulation of the expression of HAS1 and HAS2. This was associated with activation of ERK-1/2, p38, and Akt, although to a lesser extent. Both p38 and Akt were involved in CS-induced HA accumulation. IL-1beta increased HA production and levels of mRNA for HAS1, HAS2, and HAS3. CS enhanced the IL-1beta-induced level of HAS2 mRNA and reduced the level of HAS3 mRNA. IL-1beta-induced activation of p38 and JNK was slightly decreased by CS, whereas that of ERK-1/2 and Akt was enhanced. More high molecular weight HA was found in CS plus IL-1beta-treated FLS than in FLS treated with IL-1beta alone. CONCLUSION: CS stimulates the synthesis of high molecular weight HA in OA FLS through up-regulation of HAS1 and HAS2. It reduces the IL-1beta-enhanced transcription of HAS3 and increases the production of HA of large molecular sizes. These effects may be beneficial for maintaining viscosity and antiinflammatory properties in the joint.

Differential effects of hyaluronan and its fragments on fibroblasts: relation to wound healing.

Hyaluronan (HA) is involved in wound healing and its biological properties depend on its molecular size. The effects of native HA and HA-12 and HA-880 saccharide fragments on human fibroblast proliferation and expression of matrix-related genes were studied. The three HA forms promoted cell adhesion and proliferation. Matrix metalloproteinase-1 and -3 mRNA were increased by all HA forms, whereas only HA-12 stimulated the expression of the tissue inhibitor of metalloproteinase 1. HA-12 enhanced type I collagen and transforming growth factor-beta (TGF-beta) 1 expression. Interestingly, HA-12 and native HA stimulated type III collagen and TGF-beta3. HA and its fragments activated Akt and extracellular-regulated kinases 1/2 and p38. Inhibition of these signaling pathways suggested their implication in most of the effects. Only native HA activated nuclear factor-kappaB and activating protein 1. Use of CD44 siRNA suggests that this HA receptor is partly implicated in the effects, although it does not rule out the involvement of other receptors. Depending on its size, HA may exert differential regulation on the wound-healing process. Furthermore, the HA up-regulation of type III collagen and TGF-beta3 expression suggests that it may promote a fetal-like cell environment that favors scarless healing.

Interleukin-6 (IL-6) and/or soluble IL-6 receptor down-regulation of human type II collagen gene expression in articular chondrocytes requires a decrease of Sp1.Sp3 ratio and of the binding activity of both factors to the COL2A1 promoter.

Type II collagen is composed of alpha1(II) chains encoded by the COL2A1 gene. Alteration of this cartilage marker is a common feature of osteoarthritis. Interleukin-6 (IL-6) is a pro-inflammatory cytokine that needs a soluble form of receptor called sIL-6R to exert its effects in some cellular models. In that case, sIL-6R exerts agonistic action. This mechanism can make up for the partial or total absence of membrane-anchored IL-6 receptors in some cell types, such as chondrocytes. Our study shows that IL-6, sIL-6R, or both inhibit type II collagen production by rabbit articular chondrocytes through a transcriptional control. The cytokine and/or sIL-6R repress COL2A1 transcription by a -63/-35 sequence that binds Sp1.Sp3. Indeed, IL-6 and/or sIL-6R inhibit Sp1 and Sp3 expression and their binding activity to the 63-bp promoter. In chromatin immunoprecipitation experiments, IL-6.sIL-6R induced an increase in Sp3 recruitment to the detriment of Sp1. Knockdown of Sp1.Sp3 by small interference RNA and decoy strategies were found to prevent the IL-6- and/or sIL-6R-induced inhibition of COL2A1 transcription, indicating that each of these Sp proteins is required for down-regulation of the target gene and that a heterotypic Sp1.Sp3 complex is involved. Additionally, Sp1 was shown to interact with Sp3 and HDAC1. Indeed, overexpression of a full-length Sp3 cDNA blocked the Sp1 up-regulation of the 63-bp COL2A1 promoter activity, and by itself, inhibits COL2A1 transcription. We can conclude that IL-6, sIL-6R, or both in combination decrease both the Sp1.Sp3 ratio and DNA-binding activities, thus inhibiting COL2A1 transcription.

Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression.

Mesenchymal stromal cells (MSCs) seeded onto biocompatible scaffolds have been proposed for repairing bone defects. When transplanted in vivo, MSCs (expanded in vitro in 21% O(2)) undergo temporary oxygen deprivation due to the lack of pre-existing blood vessels within these scaffolds. In the present study, the effects of temporary (48 h) exposure to hypoxia (

Role of interleukin 6 (IL-6)/IL-6R-induced signal tranducers and activators of transcription and mitogen-activated protein kinase/extracellular.

OBJECTIVE: Studies have described elevated levels of interleukin 6 (IL-6) and its soluble receptor (sIL-6R) in osteoarthritic and rheumatoid joints, as well as the inhibitory effect of this combination on cartilage matrix production. We investigated the ability of IL-6/sIL-6R to modulate gene expression of matrix metalloproteinase (MMP) and ADAMTS (ADAMS with thrombospondin motifs) family members in bovine chondrocytes, and the potential role of signal transducers and activators of transcription (STAT) and mitogen-activated protein kinases (MAPK) in this regulation. METHODS: Primary cultures of bovine chondrocytes were stimulated with IL-6/sIL-6R for 30 min (Western blot and EMSA) or 24 h (RNA measurements) in the presence or absence of the STAT inhibitor parthenolide or the MAPK inhibitor PD 098059. mRNA was assessed by RT-PCR for the expression of MMP (MMP-1, -3, and -13) and 2 ADAMT family members (ADAMTS-4 and -5/11). RESULTS: IL-6/sIL-6R markedly induced activation of STAT and extracellular signal-related kinase (ERK1/2) and the subsequent expression of the collagenases MMP-1 and MMP-13 as well as MMP-3, an aggrecan-degrading enzyme and activator of pro-MMP. Expression of the 2 specific aggrecanases ADAMTS-4 and -5/11 was also elevated by this combination. Both STAT and MAPK signaling pathways were found to contribute to the IL-6/sIL-6R induction mechanisms, the overall effect being dependent on the respective magnitude of response and the crosstalk between the 2 pathways. CONCLUSION: These data indicate that the cartilage-degrading properties of IL-6/sIL-6R are mediated by induction of the aggrecan-degrading enzymes ADAMTS-4, -5/11, and MMP-3, and the collagen-degrading enzymes MMP-1 and -13. STAT and MAPK pathways play a crucial role in IL-6/sIL-6R modulation of these enzymes, suggesting that new strategies in the treatment of osteoarticular diseases might target these transduction cascades.

JAK/STAT but not ERK1/ERK2 pathway mediates interleukin (IL)-6/soluble IL-6R down-regulation of Type II collagen, aggrecan core, and link protein transcription in articular chondrocytes. Association with a down-regulation of SOX9 expression.

Signal transducers and activators of transcription (STAT) factors are cytoplasmic proteins that can be activated by Janus kinases (JAK) and that modulate gene expression in response to cytokine receptor stimulation. STAT proteins dimerize, translocate into the nucleus, and activate specific target genes. In the present study, we show for the first time that interleukin-6 (IL), in the presence of its soluble receptor (sIL-6R), induces activation of JAK1, JAK2, and STAT1/STAT3 proteins in bovine articular chondrocytes. Western blotting and mobility shift assays demonstrated that this effect is accompanied by the DNA binding of the STAT proteins. The mitogen-activated protein kinase pathway was also activated in response to IL-6/sIL-6R association, as reflected by phosphorylation of ERK1 and ERK2 proteins. In these conditions, the expression of cartilage-specific matrix genes, type II collagen, aggrecan core, and link proteins was found to be markedly down-regulated. This negative effect was abolished by addition of parthenolide, an inhibitor of the STAT activation, whereas blockade of the MAP kinases with PD098059 was without significant effect. Thus, activation of the STAT signaling pathways, but not ERK-dependent pathways, is essential for down-regulation of the major cartilage-specific matrix genes by IL-6. In addition, a parallel reduction of Sox9 expression, a key factor of chondrocyte phenotype, was found in these experimental conditions. These IL-6 effects might contribute to the phenotype loss of chondrocytes in joint diseases and the alteration of articular cartilage associated with this pathology.

Synthesis, and functional properties of a modified human insulin A-chain: implication in a 'mini-insulin' structure determination.

The design and total synthesis of a novel insulin A-chain mutant, analogue 3, is reported. In this compound, the cysteines implied in the two insulin inter-chain disulfide bridges are replaced by two serines (residues Ser(A7) and Ser(A20)) and the intra-A-chain disulfide bridge (residues Cys(A6) and Cys(A11)) is conserved. This A-chain analogue (3) has been tested in three in vitro cell culture assays, using insulin as a reference. The data clearly showed that analogue 3 mimics insulin effects on DNA synthesis, glucose uptake and glycogen synthesis without loss of potency as compared to insulin. To our knowledge, these are the first results showing that an isolated insulin chain displays functional properties similar to those of insulin. The implication of these new findings in insulin structure-function relationships and in a 'mini-insulin' structure determination is discussed.