CaMKII inhibition in human primary and pluripotent stem cell-derived chondrocytes modulates effects of TGFß and BMP through SMAD signaling.

OBJECTIVE: Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation. METHODS: Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII. RESULTS: Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in human OA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFß1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFß1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes. CONCLUSION: Current study demonstrates a direct role for CaMKII in TGF-ß and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA.

Lysophosphatidic acid mediates fibrosis in injured joints by regulating collagen type I biosynthesis.

OBJECTIVE: Articular cartilage is a highly specialized tissue which forms the surfaces in synovial joints. Full-thickness cartilage defects caused by trauma or microfracture surgery heal via the formation of fibrotic tissue characterized by a high content of collagen I (COL I) and subsequent poor mechanical properties. The goal of this study is to investigate the molecular mechanisms underlying fibrosis after joint injury. DESIGN: Rat knee joint models were used to mimic cartilage defects after acute injury. Immunohistochemistry was performed to detect proteins related to fibrosis. Human fetal chondrocytes and bone marrow stromal cells (BMSCs) were used to study the influence of the lipid lysophosphatidic acid (LPA) on COL I synthesis. Quantitative PCR, ELISA and immunohistochemistry were performed to evaluate the production of COL I. Chemical inhibitors were used to block LPA signaling both in vitro and in vivo. RESULTS: After full-thickness cartilage injury in rat knee joints, stromal cells migrating to the injury expressed high levels of the LPA-producing enzyme autotaxin (ATX); intact articular cartilage in rat and humans expressed negligible levels of ATX despite expressing the LPA receptors LPAR1 and LPAR2. LPA-induced increases in COL I production by chondrocytes and BMSCs were mediated by the MAP kinase and PI3 Kinase signaling pathways. Inhibition of the ATX/LPA axis significantly reduced COL I-enriched fibrocartilage synthesis in full-thickness cartilage defects in rats in favor of the collagen II-enriched normal state. CONCLUSION: Taken together, these results identify an attractive target for intervention in reducing the progression of post-traumatic fibrosis and osteoarthritis.

Homeobox gene Distal-less 3 (DLX3) is a regulator of villous cytotrophoblast differentiation.

Dlx3, a member of the large homeobox gene family of transcription factors, is important for murine placental development. Targeted deletion of Dlx3 in the mouse results in embryonic death due to placental failure. This study investigated the role of human DLX3 in villous cytotrophoblast (VCT) differentiation in the placenta. Primary VCT from human term placentae, which spontaneously differentiate when maintained in culture over 72 h, showed a significant increase in mRNA and protein expression of DLX3 and 3ßHSD. The functional role of DLX3 was determined using trophoblast derived-cell line, BeWo. Forskolin treated BeWo cells showed significantly increased DLX3 mRNA and protein expression. Forskolin stimulation also showed a significant increase in syncytin and 3ßHSD mRNA expression, and increased release of ßhCG into the cell culture supernatant. To determine whether DLX3 had a direct or indirect effect on VCT differentiation, mRNA and protein expression of DLX3 was increased using a plasmid DLX3 over-expression construct. Over-expression of DLX3 resulted in increased mRNA expression of 3ßHSD and syncytin, as well as increased secretion of ß-hCG protein in the cell culture medium. In conclusion, we provide evidence that DLX3 acts upstream of syncytin, 3ßHSD and ßhCG and that DLX3 has a regulatory role in VCT differentiation.

Expression, localisation and activity of ATP binding cassette (ABC) family of drug transporters in human amnion membranes.

Placental ATP-binding cassette (ABC) transporters limit fetal exposure to xenobiotics by regulating transplacental passage into the fetal circulation; their expression and function in fetal membranes, however, has not been studied. In the present study the expression, localisation and function of ABC transporters in human amnion was examined to explore their potential role in modulating amniotic fluid drug disposition in pregnancy. Single-assay oligo-microarrays were used to profile amnion gene expression, and drug transporters expressed at significant levels were identified and selected for further studies. The expression of ABCG2/breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRP) 1 (ABCC1), 2 (ABCC2) and 5 (ABCC5) was detected on the arrays, and verified by RT-PCR and immunoblotting. On confocal microscopy of fetal membrane cryosections, MRP1 and MRP5 were immunolocalised to both apical and basolateral surfaces of the amniotic epithelium, while MRP2 was expressed at low levels only in the apical membrane. BCRP in contrast showed cytoplasmic staining throughout the amniotic epithelium. In addition to the amnion, MRP1 and BCRP immunostaining was observed in the chorion and the decidua. Cell accumulation studies using selective MRP and BCRP inhibitors showed the transporters to be functionally active in amnion epithelial monolayer cultures. In contrast, transwell transport studies using intact amnion membranes did not show significant vectorial transport. These findings identify the amnion as a novel site of ABC drug transporter expression. Functional studies indicate that they may act primarily to prevent cellular xenobiotic accumulation, rather than to confer fetal protection through reduced accumulation in amniotic fluid.

The xenobiotic transporter ABCG2 plays a novel role in differentiation of trophoblast-like BeWo cells.

Trophoblast cells undergo loss of plasma membrane lipid asymmetry during cell fusion without further progression to terminal phases of apoptosis. The nature of the anti-apoptotic mechanisms providing cell survival during this process is unknown. Using a BeWo cell model, we explored the role of the xenobiotic/lipid transporter ABCG2 in promoting cell survival during forskolin-induced differentiation. Suppression of ABCG2 expression by siRNA led to a marked increase in phosphatidylserine externalisation followed by accumulation of ceramides and increased apoptosis. Expression of markers of syncytial formation (beta-hCG and HERV-W) was decreased by ABCG2 silencing, although fusion was unaffected. These findings suggest that ABCG2 protects cells during the period of transient membrane instability associated with cell differentiation and fusion, highlighting a novel, previously unrecognised role of ABCG2 as a survival factor during the formation of the placental syncytium.

Role of placenta in the regulation of fetal erythropoiesis.

We studied erythropoiesis in newborn infants delivered by mothers with normal pregnancy and gestosis. The effects of placental extracts on hemopoiesis in JCR mice were also evaluated. Our results suggest that the placenta is involved in the regulation of fetal erythropoiesis. The placenta activates fetal erythropoiesis during physiological pregnancy, while in pregnant women with gestosis the erythropoiesis-stimulating effect of placentas was less pronounced, which probably determines low reticulocyte content in the umbilical blood.