RGD peptide-induced cell death of chondrocytes and synovial cells

Background  Small peptides including the Arg-Gly-Asp (RGD) motif have been used in studies on cell-extracellular matrix (ECM) attachment due to their ability to disturb integrin-mediated attachment on the cell surface. As another biological action of RGD peptides, several reports have shown that RGD peptides are incorporated into cytoplasm and induce apoptosis by direct activation of caspase-3. This study evaluated the effect of RGD peptides on chondrocytes and synovial cells and studied the involvement of caspases. Methods  Chondrocytes and synovial cells were isolated and cultured from the knee joints of New Zealand White rabbits. Cells were incubated in serum-free medium with peptides (RGD, RGDS, GRGDSP, GRGDNP, RGES), and the survival rates were evaluated. The rate of apoptotic cells was measured by flow cytometry in cells treated with RGDS, GRGDSP, and RGES. Caspase-3, -8 and -9 activity was measured in cells treated with RGDS and GRGDSP. Osteochondral explants harvested from rabbits were also incubated with RGD peptides (RGDS, GRGDSP, and GRGDNP), and the survival rate of chondrocytes was evaluated. Results  The survival rate of cultured chondrocytes was significantly decreased in the GRGDSP- and GRGDNP-treated groups. The survival rate of synovial cells was significantly decreased with four of the RGD peptides (RGD, RGDS, GRGDSP, and GRGDNP) at 5 mM, and in the RGDS- and GRGDSP-treated groups at 1 mM. Flow cytometric assay revealed increases of apoptotic chondrocytes with GRGDSP and increases of apoptotic synovial cells with RGDS and GRGDSP. Caspase-3 was activated in chondrocytes treated with GRGDSP and it was also activated in synovial cells treated with RGDS and GRGDSP. Caspases-8 and -9 were not activated in chondrocytes or in synovial cells. The survival rate of chondrocytes in explants decreased in the superficial layer with all three RGD peptides (RGDS, GRGDSP, and GRGDNP) and in the middle layer with GRGDSP. Conclusions  RGD peptides induced apoptosis in cultured chondrocytes as well as in cells in cartilage explants and synovial cells, presumably through direct activation of caspase-3.     

Glucocorticoid induces apoptosis of osteoblast cells through the activation of glycogen synthase kinase 3β

Glucocorticoids (GCs), which play an important role in the normal regulation of bone remodeling, are widely used as anti-inflammatory and chemotherapeutic agents. However, continued exposure to GCs results in osteoporosis, which is partially due to apoptosis of osteoblasts and osteocytes. To understand the mechanism of how GCs induce cell death in osteoblasts, we examined apoptotic effects of dexamethasone (Dex), GC, on MC3T3-E1 osteoblast cells. Results revealed that Dex-induced apoptosis was inhibited by a GC receptor antagonist, mifepristone, and a general caspase inhibitor, Z-VAD-fmk, indicating that Dex induces apoptosis of MC3T3-E1 cells through the pathways involved in GC receptor and caspase. Glycogen synthase kinase 3β (GSK3β) is known to participate in apoptosis signaling in MC3T3-E1 cells. Dex activated both GSK3β and p38-mitogen-activated protein kinase (MAPK). The inhibition of GSK3β by inhibitor (LiCl) or small interference RNA (siRNA) decreased apoptosis. In contrast, the inhibition of p38-MAPK by inhibitor (SB203580) or siRNA did not decrease, but increase apoptosis. These results suggest that Dex-mediated apoptosis of osteoblasts is facilitated by GSK3β, but prevented by p38-MAPK.     

Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 micromol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 micromol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.     

Lidocaine induces apoptosis via the mitochondrial pathway independently of death receptor signaling

BACKGROUND: Local anesthetics, especially lidocaine, can lead to persistent cauda equina syndrome after spinal anesthesia. Recently, lidocaine has been reported to trigger apoptosis, although the underlying mechanisms remain unknown. To elucidate the pathway of lidocaine-induced apoptosis, the authors used genetically modified cells with overexpression or deficiencies of key regulators of apoptosis. METHODS: Human Jurkat T-lymphoma cells overexpressing the antiapoptotic protein B-cell lymphoma 2 as well as cells deficient of caspase 9, caspase 8, or Fas-associated protein with death domain were exposed to lidocaine and compared with parental cells. The authors evaluated cell viability, mitochondrial alterations, cytochrome c release, caspase activation, and early apoptosis. Apoptosis was in addition investigated in neuroblastoma cells. RESULTS: In Jurkat cells, lidocaine reduced viability, associated with a loss of the mitochondrial membrane potential. At low concentrations (3-6 mm) of lidocaine, caspase 3 was activated and release of cytochrome c was detected, whereas at higher concentrations (10 mm), no caspase activation was found. Apoptosis by lidocaine was strongly reduced by B-cell lymphoma-2 protein overexpression or caspase-9 deficiency, whereas cells lacking the death receptor pathway components caspase 8 and Fas-associated protein with death domain were not protected and displayed similar apoptotic alterations as the parental cells. Lidocaine also induced apoptotic caspase activation in neuroblastoma cells. CONCLUSIONS: Apoptosis is triggered by concentrations of lidocaine occurring intrathecally after spinal anesthesia, whereas higher concentrations induce necrosis. The data indicate that death receptors are not involved in lidocaine-induced apoptosis. In contrast, the observation that B-cell lymphoma-2 protein overexpression or the lack of caspase 9 abolished apoptosis clearly implicates the intrinsic mitochondrial death pathway in lidocaine-induced apoptosis.     

Lidocaine Induces Apoptosis via the Mitochondrial Pathway Independently of Death Receptor Signaling

Background: Local anesthetics, especially lidocaine, can lead to persistent cauda equina syndrome after spinal anesthesia. Recently, lidocaine has been reported to trigger apoptosis, although the underlying mechanisms remain unknown. To elucidate the pathway of lidocaine-induced apoptosis, the authors used genetically modified cells with overexpression or deficiencies of key regulators of apoptosis.

Methods: Human Jurkat T-lymphoma cells overexpressing the antiapoptotic protein B-cell lymphoma 2 as well as cells deficient of caspase 9, caspase 8, or Fas-associated protein with death domain were exposed to lidocaine and compared with parental cells. The authors evaluated cell viability, mitochondrial alterations, cytochrome c release, caspase activation, and early apoptosis. Apoptosis was in addition investigated in neuroblastoma cells.

Results: In Jurkat cells, lidocaine reduced viability, associated with a loss of the mitochondrial membrane potential. At low concentrations (3-6 mm) of lidocaine, caspase 3 was activated and release of cytochrome c was detected, whereas at higher concentrations (10 mm), no caspase activation was found. Apoptosis by lidocaine was strongly reduced by B-cell lymphoma-2 protein overexpression or caspase-9 deficiency, whereas cells lacking the death receptor pathway components caspase 8 and Fas-associated protein with death domain were not protected and displayed similar apoptotic alterations as the parental cells. Lidocaine also induced apoptotic caspase activation in neuroblastoma cells.     

RGD peptides immobilized on a mechanically deformable surface promote osteoblast differentiation.

The major objective of this work was to attach bone cells to a deformable surface for the effective transmission of force. We functionalized a silastic membrane and treated it with 3-aminopropyltriethoxysilane (APTS). A minimal RGD peptide was then covalently linked to the aminated surface. MC3T3-E1 osteoblast-like cells were cultured on the arginine-glycine-aspartic acid (RGD)-treated membrane for 3-15 days and cell attachment and proliferation was evaluated. We observed that cells were immediately bound to the membrane and proliferated. After 8 days on the material surface, osteoblasts exhibited high levels of ALP staining, indicating that the cells were undergoing maturation. Alizarin red staining and Fourier transform infrared (FTIR) analysis showed that the mineral formed by the cells was a biological apatite. The second objective was to apply a mechanical force to cells cultured on the modified silicone membrane. Dynamic equibiaxial strain, 2% magnitude, and a 0.25-Hz frequency were applied to bone cells for 2 h. Osteoblasts elicited increased phalloidin fluorescence, suggesting that there was reorganization of the cytoskeleton. Furthermore, the applied strain elicited increased expression of the alpha(v)beta3 integrin receptor. We concluded that the covalent binding of RGD peptides to a silicone membrane provides a compatible surface for the attachment and subsequent differentiation of osteoblasts. Moreover, the engineered surface transduces applied mechanical forces directly to the adherent cells via integrin receptors.     

Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes

Articular chondrocytes have a well-developed osmoregulatory system that enables cells to survive in a constantly changing osmotic environment. However, osmotic loading exceeding that occurring under physiological conditions severely compromises chondrocyte function and leads to degenerative changes. The aim of the present study was to investigate the form of cell death and changes in apoptotic signaling pathways under hyperosmotic stress using a primary chondrocyte culture. Cell viability and apoptosis assays performed with annexin V and propidium iodide staining showed that a highly hyperosmotic medium (600 mOsm) severely reduced chondrocyte viability and led mainly to apoptotic cell death, while elevating osmotic pressure within the physiological range caused no changes compared to isosmotic conditions. Western blot analysis revealed that a 600 mOsm hyperosmotic environment induced the activation of proapoptotic members of the mitogen-activated protein kinase family such as c-Jun N-terminal kinase (JNK) and p38, and led to an increased level of extracellular signal regulated kinase (ERK1/2). Hyperosmotic stress also induced the activation of caspase-3. In summary, our results show that hyperosmotic stress leads to mainly apoptotic cell death via the involvement of proapoptotic signaling pathways in a primary chondrocyte culture.     

Hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells: The effects of glutamate and protection by purines

Glutamate has toxic effects on a number of tissues, partly by inducing toxic (e.g., oxidative) stress, whereas adenosine can be protective. Since there is evidence that glutamate and adenosine receptors are present in bone, we set out to study whether oxidative stress, induced by hydrogen peroxide (H2O2), affected viability in the MC3T3-E1 osteoblast-like cell line and whether treatment with adenosine receptor ligands attenuated this. Hydrogen peroxide (100 microM to 5 mM) reduced the viability of the MC3T3-E1 cells, while catalase reversed this cell loss and itself had some mitogenic effect. Superoxide dismutase (SOD) increased the number of viable cells alone but failed to modify significantly the effect of H2O2 treatments. Glutamate (100 microM, 1 mM) and NMDA (10 microM), applied alone for up to 1 h, had a mitogenic effect (P < 0.05). Adenosine A1 and A2A receptor agonists and antagonists at low and high concentrations showed some mitogenic effects when added singly, but only high concentrations of the agonists showed significant protection against cell death resulting from H2O2 treatments. Contributions from both apoptotic and necrotic pathways were implicated in the H2O2-induced cell loss as was demonstrated by the use of the caspase-3 inhibitor (Z-DEVD-fmk) and the PARP-1 inhibitor (DPQ). The results demonstrate that hydrogen peroxide was toxic to MC3T3-E1 cells, whereas glutamate was not and may even have a trophic influence. Adenosine and its receptors afforded some protection to osteoblasts against cellular death mediated partly by apoptosis and partly by necrosis.     

流体剪切力抑制肿瘤坏死因子α诱导鼠成骨样细胞MC3T3-E1凋亡的实验研究

目的研究流体剪切力(fluid shear stress,FSS)对肿瘤坏死因子α(TNF-α)诱导鼠成骨样细胞MC3T3-E1凋亡的影响。方法将MC3T3-E1分为TNF-α干预组(实验组)和无TNF-α干预组(对照组),TNF-α(10ng/ml)×4h诱导MC3T3-E1凋亡后,四个实验组分别加载12dyn/cm2FSS作用0,15,30,60min。应用四甲基偶氮唑蓝(MTT)法、荧光显微镜和流式细胞技术(FΑCS)检测细胞的增殖能力和凋亡,免疫印迹法检测半胱氨酸蛋白激酶9(caspase9)和凋亡蛋白酶活化因子1(Αpaf-1)蛋白的表达。采用SPSS16.0软件包对数据进行单因素方差分析。结果TNF-α(10ng/ml)×4h能诱导明显的凋亡信号,FSS(12dyn/cm2)能明显抑制这种凋亡,而且随着刺激时间的增加,从0min逐渐延长至60min,细胞活性逐渐增加,凋亡细胞数逐渐减少,实验组与对照组单因素方差分析及各组间LSD两两比较有显著性差异(P0.05),同时caspase9和Αpaf-1蛋白的表达也逐渐增加。结论生理范围的FSS能够抑制TNF-α诱导的MC3T3-E1细胞的凋亡,作为线粒体通路的关键蛋白,caspase9和Αpaf-1在凋亡时增加而FSS后表达减少,说明FSS抑制这种凋亡至少部分是减弱了凋亡的线粒体通路。     

A synthetic peptide fragment of human MEPE stimulates new bone formation in vitro and in vivo.

Matrix extracellular phosphoglycoprotein (MEPE) was proposed as a candidate for the phosphaturic hormone phosphatonin. We found that a synthetic peptide fragment of MEPE containing the RGD and SGDG sequence stimulated new bone formation in vitro and in vivo. INTRODUCTION: Matrix extracellular phosphoglycoprotein (MEPE) was recently identified as a candidate for the phosphaturic hormone phosphatonin, which has been implicated in disturbed phosphate metabolism, rickets, and osteomalacia associated with X-linked hypophosphatemic rickets (XLH) and oncogenic hypophosphatemic osteomalacia (OHO). MEPE expression was predominantly found in osteoblasts, and mice deficient in a homolog of MEPE showed increased bone density, suggesting that MEPE produced in osteoblasts negatively regulates bone formation. In this study, we examined the effects of a synthetic 23mer peptide fragment of MEPE (AC-100, region 242-264) containing the RGD (integrin-binding) and SGDG (glycosaminoglycan-attachment) motif on bone formation in vitro and in vivo. MATERIALS AND METHODS: The osteogenic activity of AC-100 was examined in organ cultures of neonatal mouse calvariae and in vivo by injecting AC-100 onto the calvariae of mice. RESULTS: Histomorphometric examination showed that AC-100 stimulated new bone formation with increased numbers of osteoblasts in neonatal mouse calvariae in organ culture. In contrast, synthetic MEPE fragment peptides without either the RGD or SGDG motif failed to increase new bone formation. Repeated daily subcutaneous injections of AC-100 onto the calvariae in mice increased bone thickness and stimulated new bone formation as determined by the calcein double-labeling technique. However, peptides in which the RGD or SGDG sequence was scrambled did not stimulate new bone formation in vivo. AC-100 increased cell proliferation and alkaline phosphatase activity and activated focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase (ERK) in human primary osteoblasts. CONCLUSION: Our results show that a synthetic peptide corresponding with the sequence of human MEPE fragment stimulates new bone formation with increased number of osteoblasts. The results also suggest that the RGD and SGDG motifs are critical to the osteogenic activity of AC-100, presumably through activating integrin signaling pathways in osteoblasts. The anabolic effects of AC-100 may be beneficial for bone diseases associated with decreased bone formation.     

The protective role of bone morphogenetic protein-8 in the glucocorticoid-induced apoptosis on bone cells

One of the side effects associated with glucocorticoid therapy is glucocorticoid-induced bone loss. Glucocorticoids partly detain bone formation via the inhibition of osteoblastic function, however, the exact mechanism of this inhibition remains elusive. In this study, we examined the effect of dexamethasone, an active glucocorticoid analogue, on cell viability and expression of bone remodelling-related genes in primary mouse calvarial and cloned MC3T3-E1 osteoblasts. Using sensitive biochemical assays, we demonstrated the apoptotic effect of dexamethasone on osteoblastic cells. Then, utilizing Taqman probe-based quantitative RT-PCR technology, gene expression profiles of 111 bone metabolism-related genes were determined. As a result of dexamethasone treatment we have detected significant apoptotic cell death, and six genes, including Smad3, type-2 collagen α-1, type-9 collagen α-1, matrix metalloproteinase-2, bone morphogenetic protein-4 and bone morphogenetic protein-8 showed (BMP-8) significant changes in their expression on a time- and concentration-dependent manner. BMP-8, (a novel player in bone-metabolism) exhibited a two orders of magnitude elevation in its mRNA level and highly elevated protein concentration by Western blot in response to dexamethasone treatment. The knockdown of BMP-8 by RNA interference significantly increased dexamethasone-induced cell death, confirming a protective role for BMP-8 in the glucocorticoid-induced apoptosis of osteoblasts. Our results suggest that BMP-8 might be an essential player in bone metabolism, especially in response to glucocorticoids.     

Cytokines, tumor necrosis factor-alpha and interleukin-1beta, differentially regulate apoptosis in osteoarthritis cultured human chondrocytes

OBJECTIVE: This study addresses the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on cell death in human chondrocytes. METHODS: Osteoarthritis (OA) human chondrocytes stimulated with Actinomycin-D (ActD) were used as a cellular apoptotic model. Caspase family mRNA expression and protein synthesis were analyzed by the ribonuclease protection assay and Western-blot, respectively. Cell viability and apoptosis were evaluated using the 3-[4,5-dimethylthiazol-2yl] 2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Prostaglandin E2 (PGE2) and nitric oxide (NO) were evaluated by enzyme-linked immunosorbent assay (ELISA) and the Griess method, respectively. RESULTS: TNF-alpha and IL-1beta differentially affected the pattern of caspase mRNA expression by human chondrocytes. TNF-alpha induced a gradual increase in caspase-1 and -8 mRNA levels that was not seen with IL-1beta. The time sequence of caspase-3 and -7 inductions by TNF-alpha differs from that induced by IL-1beta. Cell viability was not modified by TNF-alpha or IL-1beta in cultured chondrocytes. Then, we employed ActD as a model to facilitate cell death. Treatment with TNF-alpha and ActD (TNF-alpha/ActD) increased cell death induced by ActD (23%). Treatment with IL-1beta and ActD (IL-1beta/ActD) did not modulate ActD-induced cell death. Similarly, IL-1beta/ActD did not induce an increase in the activation of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP) cleavage observed by the incubation with TNF-alpha/ActD. These different effects were not due to bcl-2 or mcl-1 levels. Inhibition of PGE2 synthesis by indomethacin increased the cell death induced by IL-1beta/Act-D (59%). An inhibitor of caspase-8 significantly reduced only the TNF-alpha/ActD-induced cell death (58%). CONCLUSION: TNF-alpha and IL-1beta differentially regulate the apoptotic pathway in human chondrocytes. This difference is dependent on PGE2 and caspase-8 levels.     

Expression and activation of peroxisome proliferator-activated receptors in growth plate chondrocytes.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a nuclear hormone receptor that is involved in a wide range of cellular processes. Although it is known that PPAR-gamma plays an important role in cell cycle control, inflammation, apoptotic cell death, and other cellular processes, the role of PPAR-gamma in the normal and pathological function of growth plate chondrocytes has not been investigated. The purpose of this study was to determine if PPARs are expressed in growth plate chondrocytes and to describe the biological effect of PPAR activation in these cells. The results demonstrate the presence of three PPAR isoforms (alpha, delta, and gamma) in growth plate cartilage. Activation of PPAR-gamma by ciglitazone in growth plate chondrocytes inhibits T(3) induced terminal differentiation and promotes apoptosis through increased levels of caspase 3/7 activity and decreased expression of the anti-apoptotic protein Bcl-2.     

Monosodium iodoacetate induces apoptosis via the mitochondrial pathway involving ROS production and caspase activation in rat chondrocytes in vitro

Monosodium iodoacetate (MIA) is an inhibitor of glyceraldehyde‐3‐phosphate dehydrogenase activity, and causes dose‐dependent cartilage degradation resembling the pathological changes of human osteoarthritis (OA). In this study, we assessed the apoptosis induced by MIA and clarified the underlying mechanisms using the primary rat chondrocytes. The apoptosis of primary rat chondrocytes was analyzed by flow cytometry. The levels of mitochondrial membrane potential (ΔΨm) were evaluated using fluorescence spectrophotometer. The production of reactive oxygen species (ROS) was determined by fluorescence spectrophotometer. Apoptosis‐related protein cytochrome c and procaspase‐3 expressions were examined by Western blotting. We found that MIA treatment induces apoptosis in chondrocytes, as confirmed by increases in the percent of apoptotic cells, up‐regulation of cytochrome c and caspase‐3 protein levels. Treatment with MIA increases ROS production and decreases the levels of ΔΨm. The antioxidant, N‐acetylcysteine (NAC), significantly prevented the production of ROS, the reduction of ΔΨm, the release of cytochrome c and the activation of caspase‐3. Further, NAC completely protected the cells from MIA‐induced apoptosis. Together these observations suggest that the mechanisms of MIA‐induced apoptosis are primarily via ROS production and mitochondria‐mediated caspase‐3 activation in primary rat chondrocytes. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 364–369, 2013     

Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro

BACKGROUND: Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. METHODS: Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3 T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti-caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti-cytochrome c Western blotting and enzyme-linked immunosorbent assays. RESULTS: Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3-like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. CONCLUSION: Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.     

Inhibition of caspase activity does not prevent the signaling phase of apoptosis in prostate cancer cells.

BACKGROUND: Caspases are a family of cysteine proteases capable of characteristically cleaving after an aspartic acid residue. Various members of the caspase family (e.g., caspases 8 and 9) have been implicated as critical initiators in the signaling phase, while others (e.g., caspases 3, 6, and 7) have been implicated in the effector or execution phase of apoptosis. Thapsigargin (TG) is capable of inducing cell proliferation-independent apoptosis of prostate cancer cells. This study was undertaken to determine if caspase inhibition can prevent TG- or 5-fluorodeoxyuridine (5-FrdU)-induced apoptosis in prostate cancer cells. METHODS: Caspase activity was evaluated by Western blot analysis of the cleavage of retinoblastoma (Rb) protein, a caspase substrate during TG-induced death of prostate cancer cells. In addition, hydrolysis of caspase-specific fluorescent peptide substrates was assayed in lysates from TG-treated cells. Clonogenic survival assays were performed following treatment of rat AT3 and human TSU-Pr1 prostate cancer cell lines with TG and 5-FrdU in the presence and absence of peptide caspase inhibitors. AT3.1 cells transfected with the crmA gene, encoding a viral protein with caspase-inhibitory activity, were also tested for clonogenic survival following TG and 5-FrdU exposure. RESULTS: During treatment with TG, Rb is first dephosphorylated and then proteolytically cleaved into 100-kDa and 40-kDa forms, indicative of caspase activity. A 6-8-fold increase in class II (i.e., caspases 3, 7, and 10) hydrolysis of the caspase substrate Z-DEVD-AFC was observed after 24 hr of TG or 5-FrdU. AT3 cells expressing crmA (i.e., an inhibitor of caspases 1, 4, and 8) were not protected from apoptosis induced by TG or 5-FrdU. The caspase inhibitors Z-DEVD-fmk (i.e., an inhibitor of caspases 3, 7, and 10) and Z-VAD-fmk (i.e., a general caspase inhibitor) were also unable to protect TSU and AT3 cells from apoptosis induced by TG or 5-FrdU. CONCLUSIONS: Caspase activation may play a role in the downstream effector phase of the apoptotic cascade; however, in this study, caspase inhibition did not prevent the signaling phase of apoptosis induced by two agents with distinct mechanisms of cytotoxicity, TG or 5-FrdU. These results suggest that caspase inhibition by recently described endogenous caspase inhibitors should not lead to development of resistance to TG. A strategy for targeting TG's unique cytotoxicity to metastatic prostate cancer cells is currently under development.     

Fidgetin-like 1 gene inhibited by basic fibroblast growth factor regulates the proliferation and differentiation of osteoblasts.

The FIGNL1 gene was proven to be a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). In this in vitro study, the AAA proteins inhibited osteoblast proliferation and stimulated osteoblast differentiation. We showed that FIGNL1 may play some regulatory role in osteoblastogenesis. INTRODUCTION: The fidgetin-like 1 (FIGNL1) gene encodes a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). Although the FIGNL1 protein localizes to both the nucleus and cytoplasm, the function of FIGNL1 remains unknown. In a previous study, we identified several genes that mediate the anabolic effects of basic fibroblast growth factor (bFGF) on bone by using microarray data. FIGNL1 was one of the genes that downregulated >2-fold in MC3T3-E1 cells after treatment with bFGF. Therefore, this study was aimed to identify and confirm the function of FIGNL1 on osteoblastogenesis. MATERIALS AND METHODS: We examined the effect of the FIGNL1 gene on proliferation, differentiation, and apoptosis in mouse osteoblast cells (MC3T3-E1 and mouse primary calvarial cells) using flow cytometry, RT-PCR, cell proliferation assay, and cell death assay. MC3T3-E1 cells and mouse calvarial cells were transfected with small interfering RNA (siRNA) directed against the FIGNL1 or nontargeting control siRNA and examined by cell proliferation and cell death assays. Also, FIGNL1 was fused to enhance green fluorescent protein (EGFP), and the EGFP-fused protein was transiently expressed in MC3T3-E1 cells. RESULTS: Reduced expression of FIGNL1 by bFGF and TGF-beta1 treatment was verified by RT-PCR analysis. Overexpression of FIGNL1 reduced the proliferation of MC3T3-E1 and calvarial cells, more than the mock transfected control cells did. In contrast, siFIGNL1 transfection significantly increased the proliferation of osteoblasts, whereas overexpression of FIGNL1 did not seem to alter apoptosis in osteoblasts. Meanwhile, overexpression of FIGNL1 enhanced the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in osteoblasts. In contrast, siFIGNL1 decreased the expression of ALP and OCN. A pEGFP-FIGNL1 transfected into MCT3-E1 cells had an initially ubiquitous distribution and rapidly translocated to the nucleus 1 h after bFGF treatment. CONCLUSIONS: From these results, we proposed that FIGNL1, a subfamily member of the AAA family of proteins, might play some regulatory role in osteoblast proliferation and differentiation. Further analyses of FIGNL1 will be needed to better delineate the mechanisms contributing to the inhibition of proliferation and stimulation of osteoblast differentiation.     

Caspase-dependent cleavage of cadherins and catenins during osteoblast apoptosis.

As transmembrane, Ca2+-dependent cell-cell adhesion molecules, cadherins play a central role in tissue morphogenesis and homeostasis. Stable adhesion is dependent on interactions of the cytoplasmic domain of the cadherins with a group of intracellular proteins, the catenins. In the present study, we have detected the expression of alpha-, beta-, and gamma-catenins in human osteoblasts, which assemble with cadherins to form two distinct complexes containing cadherin and alpha-catenin, with either beta- or gamma-catenin. In osteoblasts undergoing apoptosis, proteolytic cleavage of N-cadherin and beta- and gamma- catenins but not alpha-catenin was associated with the activation of caspase-3 and prevented by the caspase inhibitor Z-VAD-fmk. The pattern of cadherin/catenin cleavage detected in apoptotic osteoblasts was reproduced in vitro by recombinant caspase-3. The presence of a 90-kDa extracellular domain fragment of N-cadherin in conditioned medium from apoptotic cells indicates that additional extracellular or membrane-associated proteases also are activated. Disruption of N-cadherin-mediated cell-cell adhesion with function-blocking antibodies induced osteoblast apoptosis, activation of caspases, and cleavage of beta-catenin. These findings provide compelling evidence that N-cadherin-mediated cell-cell adhesion promotes osteoblast survival and suggest that the underlying mechanism may involve activation of beta-catenin signaling.