The oncofetal gene survivin is re-expressed in osteoarthritis and is required for chondrocyte proliferation in vitro.

BACKGROUND: Regulation of cell death and cell division are key processes during chondrogenesis and in cartilage homeostasis and pathology. The oncogene survivin is considered to be critical for the coordination of mitosis and maintenance of cell viability during embryonic development and in cancer, and is not detectable in most adult differentiated tissues and cells. We analyzed survivin expression in osteoarthritic cartilage and its function in primary human chondrocytes in vitro. METHODS: Survivin expression was analyzed by immunoblotting and quantitative real-time PCR. The localization was visualized by immunofluorescence. Survivin functions in vitro were investigated by transfection of a specific siRNA. RESULTS: Survivin was expressed in human osteoarthritic cartilage, but was not detectable in macroscopically and microscopically unaffected cartilage of osteoarthritic knee joints. In primary human chondrocyte cultures, survivin was localized to heterogeneous subcellular compartments. Suppression of survivin resulted in inhibition of cell cycle progression and sensitization toward apoptotic stimuli in vitro. CONCLUSIONS: The present study indicates a role for survivin in osteoarthritic cartilage and human chondrocytes. In vitro experiments indicated its involvement in cellular division and viability. Learning more about the functions of survivin in chondrocyte biology might further help toward understanding and modulating the complex processes of cartilage pathology and regeneration.

The oncofetal gene survivin promotes cell proliferation and survival in primary human osteoblastic cells.

Survivin, the smallest member of the inhibitor of apoptosis gene family, is critical for the regulation of mitosis and maintenance of cell viability during embryonic development and cancer, while not being detectable in most adult differentiated tissues. We know little about whether survivin plays any physiological or pathophysiological role in the adult musculoskeletal system. We studied the expression of survivin in primary human osteoblastic cells and its biological functions in vitro. Survivin was detected by immunoblotting and real-time PCR. Subcellular localization was analyzed by immunofluorescence. Transfection of siRNA and plasmids coding for wild-type survivin was performed to study survivin function, i.e., proliferation and apoptosis assays. Survivin mRNA and protein are expressed in primary human osteoblastic cells. During interphase survivin localizes predominantly to the cytoplasmic compartment, which is relevant for the organization of the spindle apparatus during mitosis. Survivin knockdown resulted in an arrest of the cell cycle at the G(2)/M phase and increased rates of apoptosis. Elevated levels of survivin in primary human osteoblasts enhanced proliferation and cell viability. Taken together, we demonstrate for the first time that survivin is expressed in primary human osteoblastic cells on the mRNA and protein levels. Our results indicate that survivin is a critical factor for cell division and cell viability in primary human osteoblastic cells. Learning more about survivin's role in human osteoblasts could be an important step toward understanding the complex processes involved in bone homeostasis and remodeling.

The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro.

BACKGROUND: Chondrosarcoma is virtually resistant to chemotherapy and radiation therapy. Survivin, the smallest member of the inhibitor of apoptosis protein family, is a critical factor for tumor progression and resistance to conventional therapeutic approaches in a wide range of malignancies. However, the role of survivin in chondrosarcoma has not been well studied. We examined the importance of survivin gene expression in chondrosarcoma and analysed its influences on proliferation, apoptosis and resistance to chemotherapy in vitro. METHODS: Resected chondrosarcoma specimens from which paraffin-embedded tissues could be extracted were available from 12 patients. In vitro experiments were performed in human chondrosarcoma cell lines SW1353 and Hs819.T. Immunohistochemistry, immunoblot, quantitative PCR, RNA interference, gene-overexpression and analyses of cell proliferation and apoptosis were performed. RESULTS: Expression of survivin protein was detected in all chondrosarcoma specimens analyzed, while undetectable in adult human cartilage. RNA interference targeting survivin resulted in a G2/M-arrest of the cell cycle and led to increased rates of apoptosis in chondrosarcoma cells in vitro. Overexpression of survivin resulted in pronounced resistance to doxorubicin treatment. CONCLUSIONS: These findings indicate that survivin plays a role in the pathogenesis and pronounced chemoresistance of high grade chondrosarcoma. Survivin antagonizing therapeutic strategies may lead to new treatment options in unresectable and metastasized chondrosarcoma.

Requirement for class II phosphoinositide 3-kinase C2alpha in maintenance of glomerular structure and function.

An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.

Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2beta to the nucleus.

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2alpha and PI3K-C2beta act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pools of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2beta translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2beta resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme, firstly in the cytosol and secondly in the nuclei. Using detergent-treated nuclei, we showed that PI3K-C2beta co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically, and a phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2beta levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutations of PI3K-C2beta demonstrated that epidermal growth factor-driven translocation to the nucleus is dependent on a sequence of basic amino acid residues (KxKxK) that form a nuclear localization motif within the C-terminal C2 domain. Furthermore, when this sequence was expressed as an EGFP (enhanced green fluorescent protein) fusion protein, it translocated fluorescence into nuclei with an efficiency dependent upon copy number. These data demonstrate that epidermal growth factor stimulates the appearance of PI3K-C2beta in nuclei. Further, this effect is dependent on a nuclear localization signal present within the C-terminal C2 domain, indicating its bimodal function regulating phospholipid binding and shuttling PI3K-C2beta into the nucleus.