Endogenous IGFBP‐3 is required for both growth factor‐stimulated cell proliferation and cytokine‐induced apoptosis in mammary epithelial cells

TNF‐α and IGF‐I exert opposing effects on mammary epithelial cell (MEC) growth and survival. However, both increase IGF binding protein‐3 (IGFBP‐3) expression, a multifunctional protein that plays both IGF‐dependent as well as independent roles in these processes. We have reported that IGF‐I utilizes the PI3‐K and MAPK pathways to induce IGFBP‐3 expression in bovine MEC. Here we show that TNF‐α requires the SAPK pathway p38, but not JNK, to induce IGFBP‐3 expression. Contrary to reports in cancer cell lines, TNF‐α retained its ability to decrease DNA synthesis in cells transfected with IGFBP‐3 siRNA. It also retained its ability to inhibit IGF‐I‐stimulated DNA synthesis in these cells. In contrast, the ability of IGF‐I to increase DNA synthesis was attenuated with IGFBP‐3 knockdown. IGFBP‐3 knockdown also decreased basal DNA synthesis, indicating that a certain level of IGFBP‐3 may be required for cell proliferation. While TNF‐α alone failed to induce apoptosis, it increased cell death when added with the JNK agonist anisomycin (ANS). TNF‐α and ANS were unable to induce apoptosis when either IGFBP‐3 or JNK‐2 was knocked‐down, suggesting that both JNK and IGFBP‐3 may interact with a downstream molecule central to apoptosis. There are reports that IGFBP‐3 promotes either cell proliferation or apoptosis in different cell systems. However, this is the first report that endogenous IGFBP‐3 is required for the action of both stimulatory and inhibitory factors within the same cell line. Therefore, the actions of IGFBP‐3 are not pre‐determined, but instead governed by cellular context such as JNK activation. J. Cell. Physiol. 220: 182–188, 2009. © 2009 Wiley‐Liss, Inc.     

Insulin‐like growth factor‐I prevents caspase‐mediated apoptosis in Schwann cells

Both neurons and glia succumb to programmed cell death (PCD) when deprived of growth factors at critical periods in development or following injury. Insulin‐like growth factor‐I (IGF‐I) prevents apoptosis in neurons in vitro. To investigate whether IGF‐I can protect Schwann cells (SC) from apoptosis, SC were harvested from postnatal day 3 rats and maintained in serum‐containing media until confluency. When cells were switched to serum‐free defined media (DM) for 12–72 h, they underwent PCD. Addition of insulin or IGF‐I prevented apoptosis. Bisbenzamide staining revealed nuclear condensation and formation of apoptotic bodies in SC grown in DM alone, but SC grown in DM plus IGF‐I had normal nuclear morphology. The phosphatidylinositol 3‐kinase (PI 3‐K) inhibitor LY294002 blocked IGF‐I–mediated protection. Caspase‐3 activity was rapidly activated upon serum withdrawal in SC, and the caspase inhibitor BAF blocked apoptosis. These results suggest that IGF‐I rescues SC from apoptosis via PI 3‐K signaling which is upstream from caspase activation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 540–548, 1999     

Differential insulin‐like growth factor (IGF)‐independent interactions of IGF binding protein‐3 and IGF binding protein‐5 on apoptosis in human breast cancer cells. Involvement of the mitochondria.

We have demonstrated previously in Hs578T cells that insulin‐like growth factor binding protein (IGFBP)‐3 can significantly accentuate ceramide (C2)‐induced apoptosis, but has no effect on cell death induced by integrin detachment [using an arginine‐glycine‐aspartic acid (RGD)‐containing peptide]. In contrast we found that IGFBP‐5 could inhibit apoptosis induced by either C2 or integrin detachment. It is now clear that the mitochondria not only provide the energy required for cell viability, but can also play an important role during the commitment phase to apoptosis. We used a mitochondrial respiratory chain inhibitor, antimycin A, at both apoptotic and nonapoptotic doses to further investigate the IGF‐independent actions of IGFBP‐3 and IGFBP‐5 on C2 and RGD‐induced apoptosis in the Hs578T cells. Hs578T cells had one of three treatments. 1: They were incubated with increasing doses of antimycin A for 24 h. 2: They were coincubated with an apoptotic dose of either C2 or RGD together with a nonapoptotic dose of antimycin A for 24 h. 3: They were incubated with a binding protein (100 ng/ml) for 24 h followed by coincubation of the binding protein with an apoptotic dose of antimycin A for a further 24 h. Cell viability was assessed by trypan blue dye exclusion and MTT assay, and apoptosis was confirmed and measured by morphologic assessment and flow cytometry. We found that antimycin A initiated apoptosis at 10 μmol/L and above. We also demonstrated that a nonapoptotic dose of antimycin A (0.1 μmol/L) significantly inhibited C2‐induced apoptosis, whereas it significantly accentuated RGD‐induced cell death. In addition, we found that cell death induced by antimycin A can be accentuated by IGFBP‐3 but is not affected by IGFBP‐5. These data indicate that IGFBP‐3 can directly enhance apoptosis triggered via the mitochondria; either directly by a mitochondrial inhibitor or by C2 (which we demonstrate to act via effects on the mitochondria in this model). IGFBP‐5, however, appears to confer survival effects via a distinct pathway not involving the mitochondria. J. Cell. Biochem. 80:248–258, 2000. © 2000 Wiley‐Liss, Inc.     

Polymerization of insulin‐like growth factor‐binding protein‐1 (IGFBP‐1) potentiates IGF‐I actions in placenta

Insulin‐like growth factor (IGF)‐binding protein‐1 (IGFBP‐1), the main secretory protein of decidua that binds to IGFs and has been shown to inhibit or stimulate IGFs' bioactivities. Polymerization, one of the posttranslational modifications of IGFBP‐1, has been shown to lead to loss of inhibiting effect of IGFBP‐1 on IGF‐I actions. The current studies were undertaken to elucidate the effects of steroid hormones on IGFBP‐1 polymerization in trophoblast cell cultures. Placental tissues were obtained during legal, elective procedures of termination of pregnancy performed between 7 and 10 weeks of gestation, and primary trophoblast cells were separated. IGFBP‐1 polymerization was analyzed by SDS–PAGE and immunoblotting. IGFBP‐1 was polymerized when IGFBP‐1 was added to trophoblast cell cultures. Polymerization of IGFBP‐1 was inhibited by the addition of anti‐tissue transglutaminase antibody into the culture media. There was an increase in the intensity of polymerized IGFBP‐1 bands with the addition of medroxyprogesterone acetate (MPA), while no such difference was observed upon treatment with estradiol. MPA also increased the expression of tissue transglutaminase on trophoblast cell membranes. IGF‐I stimulated trophoblast cell migration, while IGFBP‐1 inhibited this IGF‐I‐induced trophoblast response. Addition of MPA attenuated the inhibitory effects of IGFBP‐1 on IGF‐I‐induced trophoblast cell migration. IGFBP‐1 was polymerized by tissue transglutaminase on the cell surface of trophoblasts, and MPA increased tissue transglutaminase expression on the cell surface and facilitated IGFBP‐1 polymerization. These results suggest that progesterone might facilitate polymerization of decidua‐secreted IGFBP‐1 and increase IGF‐I actions at feto‐maternal interface, thereby stimulating trophoblast invasion of maternal uterus. J. Cell. Physiol. 226: 434–439, 2011. © 2010 Wiley‐Liss, Inc.     

Testes‐specific transgene expression in insulin‐like growth factor‐I transgenic mice

Insulin‐like growth factor‐I (IGF‐I) is a low molecular weight peptide that mediates the cell proliferating actions of growth hormone. Evidence exists indicating that IGF‐I is produced by various cell types and this growth factor has been implicated in a variety of reproductive processes. To investigate the effect of IGF‐I over‐expression on reproductive systems, we generated three independent lines of transgenic mice harbouring a human IGF‐I cDNA (hIGF‐I) under the control of a Cytomegalovirus immediate early (CMV) promoter. The CMV promoter was used in an attempt to direct expression of IGF‐I into a variety of tissues both reproductive and non‐reproductive. Yet expression of the foreign hIGF‐I gene, determined by Northern blot, was found to occur only in the testicular tissues of the male mice, apparently due to methylation of the transgene in all the tissues tested except the testes, which demonstrate transgene hypomethylation. Evaluation of the transgene expression during testicular development revealed that expression begins between 10 and 15 days of development, coinciding with the appearance of the zygotene and pachytene primary spermatocytes during early spermatogenesis, therefore indicating germ line expression of the transgene. Extensive study of the CMV‐hIGF‐I transgenic lines of mice has revealed that the effects of the transgene expression do not extend beyond the testicular tissues. No significant differences (P > 0.05) in the IGF‐I serum levels, growth rates, or testicular histology have been observed between transgenic and non‐transgenic male siblings. The ability of transgenic males to produce offspring also appears unaffected. Evaluation of the IGF binding protein (IGFBP) levels in the testicular tissues of CMV‐hIGF‐I transgenic mice by Western ligand blot revealed an increase in the concentration of testicular proteins with molecular weights corresponding to IGFBP‐2 and IGFBP‐3. These results suggest that the testicular over‐expression of IGF‐I induces increased IGFBP localization in this tissue. Inhibition of IGF activity by the IGFBPs would explain the lack of a dramatic physiological effect in the CMV‐hIGF‐I transgenic mice, despite the presence of elevated testicular IGF‐I. The observation that testis specific IGF‐I overexpression induces localization of IGFBPs in this tissue confirms the existence of a well regulated testicular IGF system and supports the convention that this growth factor plays an important role in testicular function. Mol. Reprod. Dev. 54:32–42, 1999. © 1999 Wiley‐Liss, Inc.     

Regulation of Survivin and Bcl‐2 in HepG2 Cell Apoptosis Induced by Quercetin

Quercetin, a widely distributed bioflavonoid, has been shown to induce growth inhibition in a variety of human cancer cells. However, the regulation of survivin and Bcl‐2 on the quercetin‐induced cell‐growth inhibition and apoptosis in cancer cells remains unclear. In the present study, we report that quercetin can inhibit proliferation and induce apoptosis in HepG2 cells in dose‐ and time‐dependent manner. Hoechst 33258 and acridine orange/ethidium bromide (AO/EB) staining showed that HepG2 cells underwent the typical morphologic changes of apoptosis characterized by nuclear shrinkage, chromatin condensation, or fragmentation after exposure to quercetin. Cell‐cycle analysis reveals a significant increase of the proportion of cells in G₀/G₁ phase. We also demonstrate that the levels of survivin and Bcl‐2 protein expression in HepG2 cells decreased concurrently, and the levels of p53 protein increased significantly after treatment with quercetin by immunocytochemistry analysis. Relative activity of caspase‐3 and caspase‐9 increased significantly. These data clearly indicate that quercetin‐induced apoptosis is associated with caspase activation, and the levels of survivin and Bcl‐2. Our results indicate that the expression of survivin may be associated with Bcl‐2 expression, and the inhibition expression of survivin, in conjunction with Bcl‐2, might cause more pronounced apoptotic effects. Together, concurrent down‐regulated survivin and Bcl‐2 play an important role in HepG2 cell apoptosis induced by quercetin.     

Down‐regulation of insulin‐like growth factor binding protein 5 is involved in intervertebral disc degeneration via the ERK signalling pathway

It is obvious that epigenetic processes influence the evolution of intervertebral disc degeneration (IDD). However, its molecular mechanisms are poorly understood. Therefore, we tested the hypothesis that IGFBP5, a potential regulator of IDD, modulates IDD via the ERK signalling pathway. We showed that IGFBP5 mRNA was significantly down‐regulated in degenerative nucleus pulposus (NP) tissues. IGFBP5 was shown to significantly promote NP cell proliferation and inhibit apoptosis in vitro, which was confirmed by MTT, flow cytometry and colony formation assays. Furthermore, IGFBP5 was shown to exert its effects by inhibiting the ERK signalling pathway. The effects induced by IGFBP5 overexpression on NP cells were similar to those induced by treatment with an ERK pathway inhibitor (PD98059). Moreover, qRT‐PCR and Western blot analyses were performed to examine the levels of apoptosis‐related factors, including Bax, caspase‐3 and Bcl2. The silencing of IGFBP5 up‐regulated the levels of Bax and caspase‐3 and down‐regulated the level of Bcl2, thereby contributing to the development of human IDD. Furthermore, these results were confirmed in vivo using an IDD rat model, which showed that the induction of Igfbp5 mRNA expression abrogated the effects of IGFBP5 silencing on intervertebral discs. Overall, our findings elucidate the role of IGFBP5 in the pathogenesis of IDD and provide a potential novel therapeutic target for IDD.     

RNAi‐mediated silencing of insulin receptor substrate‐4 enhances actinomycin D‐ and tumor necrosis factor‐α‐induced cell death in hepatocarcinoma cancer cell lines

Insulin receptor substrate‐4 (IRS‐4) transmits signals from the insulin‐like growth factor receptor (IGF‐IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS‐4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS‐4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS‐4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor‐α (TNF‐α). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial‐dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF‐α amplifies the effect of Act D on HepG2 cell apoptosis increasing c‐jun N‐terminal kinase (JNK) activity, IκB‐α proteolysis and glutathione depletion. IRS‐4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS‐4 action is independent of Akt, IκB kinase and JNK. IRS‐4 down regulation, however, decreased γ‐glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF‐α. These results suggest that IRS‐4 protects HepG2 cells from oxidative stress induced by drug treatment. J. Cell. Biochem. 108: 1292–1301, 2009. © 2009 Wiley‐Liss, Inc.     

Reactive oxygen species‐induced cell death of rat primary astrocytes through mitochondria‐mediated mechanism

Astrocytes, the most abundant glial cell population in the central nervous system (CNS), play physiological roles in neuronal activities. Oxidative insult induced by the injury to the CNS causes neural cell death through extrinsic and intrinsic pathways. This study reports that reactive oxygen species (ROS) generated by exposure to the strong oxidizing agent, hexavalent chromium (Cr(VI)) as a chemical‐induced oxidative stress model, caused astrocytes to undergo an apoptosis‐like cell death through a caspase‐3‐independent mechanism. Although activating protein‐1 (AP‐1) and NF‐κB were activated in Cr(VI)‐primed astrocytes, the inhibition of their activity failed to increase astrocytic cell survival. The results further indicated that the reduction in mitochondrial membrane potential (MMP) was accompanied by an increase in the levels of ROS in Cr(VI)‐primed astrocytes. Moreover, pretreatment of astrocytes with N‐acetylcysteine (NAC), the potent ROS scavenger, attenuated ROS production and MMP loss in Cr(VI)‐primed astrocytes, and significantly increased the survival of astrocytes, implying that the elevated ROS disrupted the mitochondrial function to result in the reduction of astrocytic cell viability. In addition, the nuclear expression of apoptosis‐inducing factor (AIF) and endonuclease G (EndoG) was observed in Cr(VI)‐primed astrocytes. Taken together, evidence shows that astrocytic cell death occurs by ROS‐induced oxidative insult through a caspase‐3‐independent apoptotic mechanism involving the loss of MMP and an increase in the nuclear levels of mitochondrial pro‐apoptosis proteins (AIF/EndoG). This mitochondria‐mediated but caspase‐3‐independent apoptotic pathway may be involved in oxidative stress‐induced astrocytic cell death in the injured CNS. J. Cell. Biochem. 107: 933–943, 2009. © 2009 Wiley‐Liss, Inc.     

No Decrease in Free IGF‐I with Increasing Insulin in Obesity‐Related Insulin Resistance

Objective: Different facts suggest that the insulin growth factor (IGF)/ insulin growth factor‐binding protein (IGFBP) system may be regulated by factors other than growth hormone. It has been proposed that, in healthy subjects, free IGF‐I plays a role in glucose metabolism. The role of free IGF‐I in glucose homeostasis in insulin resistance is poorly understood. This study was undertaken to evaluate the effects of acute changes in plasma glucose and insulin levels on free IGF‐I and IGFBP‐1 in obese and non‐obese subjects. Research Methods and Procedures: Nineteen lean and 24 obese subjects were investigated. A frequently sampled intravenous glucose tolerance test was performed. Free IGF‐I and IGFBP‐1 were determined at 0, 19, 22, 50, 100, and 180 minutes. Results: Basal free IGF‐I levels tended to be higher and IGFBP‐1 lower in obese than in lean subjects. IGFBP‐1 levels inversely correlated with basal insulin concentration. To determine the effects of insulin on the availability of free IGF‐I and IGFBP‐1, changes in their plasma concentrations were measured during a frequently sampled intravenous glucose tolerance test. After insulin administration, a significant suppression of free IGF‐I at 22% was observed in lean subjects. In contrast, plasma‐free IGF‐I levels remained essentially unchanged in the obese group. The differences between both groups were statistically significant at 100 minutes (p < 0.01) and 180 minutes (p < 0.05). Serum IGFBP‐1 was suppressed to a similar extent in both groups. Discussion: These data suggest that the concentrations of free IGF‐I and IGFBP‐1 are differentially regulated by obesity. Obesity‐related insulin resistance leads to unsuppressed free IGF‐I levels.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Deficiency of insulin‐like growth factor 1 reduces vulnerability to chronic alcohol intake‐induced cardiomyocyte mechanical dysfunction: role of AMPK

Circulating insulin‐like growth factor I (IGF‐1) levels are closely associated with cardiac performance although the role of IGF‐1 in alcoholic cardiac dysfunction is unknown. This study was designed to evaluate the impact of severe liver IGF‐1 deficiency (LID) on chronic alcohol‐induced cardiomyocyte contractile and intracellular Ca²⁺ dysfunction. Adult male C57 and LID mice were placed on a 4% alcohol diet for 15 weeks. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time‐to‐relengthening (TR₉₀), change in fura‐fluorescence intensity (ΔFFI) and intracellular Ca²⁺ decay. Levels of apoptotic regulators caspase‐3, Bcl‐2 and c‐Jun NH2‐terminal kinase (JNK), the ethanol metabolizing enzyme mitochondrial aldehyde dehydrogenase (ALDH2), as well as the cellular fuel gauge AMP‐activated protein kinase (AMPK) were evaluated. Chronic alcohol intake enlarged myocyte cross‐sectional area, reduced PS, ± dL/dt and ΔFFI as well as prolonged TR₉₀ and intracellular Ca²⁺ decay, the effect of which was greatly attenuated by IGF‐1 deficiency. The beneficial effect of LID against alcoholic cardiac mechanical defect was ablated by IGF‐1 replenishment. Alcohol intake increased caspase‐3 activity/expression although it down‐regulated Bcl‐2, ALDH2 and pAMPK without affecting JNK and AMPK. IGF‐1 deficiency attenuated alcoholism‐induced responses in all these proteins with the exception of Bcl‐2. In addition, the AMPK agonist 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside abrogated short‐term ethanol incubation‐elicited cardiac mechanical dysfunction. Taken together, these data suggested that IGF‐1 deficiency may reduce the sensitivity to ethanol‐induced myocardial mechanical dysfunction. Our data further depicted a likely role of Caspase‐3, ALDH2 and AMPK activation in IGF‐1 deficiency induced ‘desensitization’ of alcoholic cardiomyopathy.     

Cross‐talk between MAP kinase pathways is involved in IGF‐independent,IGFBP‐6‐induced Rh30 rhabdomyosarcoma cell migration

Insulin‐like growth factor binding protein‐6 (IGFBP‐6) inhibits the tumorigenic properties of IGF‐II‐dependent cancer cells by directly inhibiting IGF‐II actions. However, in some cases, IGFBP‐6 is associated with increased cancer cell tumorigenicity, which is unlikely to be due to IGF‐II inhibition. The mechanisms underlying the contradictory actions of IGFBP‐6 remain unclear. We recently generated an IGFBP‐6 mutant that does not bind IGFs (mIGFBP‐6) to address this issue. Although RD rhabdomyosarcoma cells express IGF‐II, we previously showed that mIGFBP‐6 promoted migration through an IGF‐independent, p38‐dependent pathway. We further studied the role of MAP kinases in IGFBP‐6‐induced migration of Rh30 rhabdomyosarcoma cells, which also express IGF‐II. In these cells, mIGFBP‐6 induced chemotaxis rather than chemokinesis. Both wild‐type (wt) and mIGFBP‐6 transiently induced phosphorylation of ERK1/2 and JNK1, but not p38. Inhibition of ERK1/2 phosphorylation completely prevented mIGFBP‐6‐induced ERK1/2 activation and cell migration, whereas a JNK inhibitor partially prevented migration. Interestingly, p38 pathway inhibition completely prevented mIGFBP‐6‐induced ERK1/2 and JNK1 activation and migration despite mIGFBP‐6 not activating p38. Furthermore, blocking the ERK1/2 pathway also inhibited mIGFBP‐6‐induced JNK1 activation. In contrast, IGFBP‐6 had no effect on Akt phosphorylation and an Akt inhibitor had no effect on migration. These results indicate that IGFBP‐6 promotes Rh30 rhabdomyosarcoma chemotaxis in an IGF‐independent manner, and that MAPK signaling pathways and their cross‐talk play an important role in this process. Therefore, besides decreasing Rh30 cell proliferation by inhibiting IGF‐II, IGFBP‐6 promotes their migration via a distinct pathway. Understanding these disparate actions of IGFBP‐6 may lead to the development of novel cancer therapeutics. J. Cell. Physiol. 224: 636–643, 2010. © 2010 Wiley‐Liss, Inc.     

Cross‐talk between IGF‐1 and estrogen receptors attenuates intracellular changes in ventral spinal cord 4.1 motoneuron cells because of interferon‐gamma exposure

Insulin‐like growth factor‐1 (IGF‐1) is a neuroprotective growth factor that promotes neuronal survival by inhibition of apoptosis. To examine whether IGF‐1 exerts cytoprotective effects against extracellular inflammatory stimulation, ventral spinal cord 4.1 (VSC4.1) motoneuron cells were treated with interferon‐gamma (IFN‐γ). Our data demonstrated apoptotic changes, increased calpain:calpastatin and Bax:Bcl‐2 ratios, and expression of apoptosis‐related proteases (caspase‐3 and ‐12) in motoneurons rendered by IFN‐γ in a dose‐dependent manner. Post‐treatment with IGF‐1 attenuated these changes. In addition, IGF‐1 treatment of motoneurons exposed to IFN‐γ decreased expression of inflammatory markers (cyclooxygenase‐2 and nuclear factor‐kappa B:inhibitor of kappa B ratio). Furthermore, IGF‐1 attenuated the loss of expression of IGF‐1 receptors (IGF‐1Rα and IGF‐1Rβ) and estrogen receptors (ERα and ERβ) induced by IFN‐γ. To determine whether the protective effects of IGF‐1 are associated with ERs, ERs antagonist ICI and selective siRNA targeted against ERα and ERβ were used in VSC4.1 motoneurons. Distinctive morphological changes were observed following siRNA knockdown of ERα and ERβ. In particular, apoptotic cell death assessed by TUNEL assay was enhanced in both ERα and ERβ‐silenced VSC4.1 motoneurons following IFN‐γ and IGF‐1 exposure. These results suggest that IGF‐1 protects motoneurons from inflammatory insult by a mechanism involving pivotal interactions with ERα and ERβ.

     

Synthetic polymeric substrates as potent pro‐oxidant versus anti‐oxidant regulators of cytoskeletal remodeling and cell apoptosis

The role of reactive oxygen species (ROS)‐mediated cell signal transduction pathways emanating from engineered cell substrates remains unclear. To elucidate the role, polymers derived from the amino acid L ‐tyrosine were used as synthetic matrix substrates. Variations in their chemical properties were created by co‐polymerizing hydrophobic L ‐tyrosine derivatives with uncharged hydrophilic poly(ethylene glycol) (PEG, Mw = 1,000 Da), and negatively charged desaminotyrosyl‐tyrosine (DT). These substrates were characterized for their intrinsic ability to generate ROS, as well as their ability to elicit Saos‐2 cell responses in terms of intracellular ROS production, actin remodeling, and apoptosis. PEG‐containing substrates induced both exogenous and intracellular ROS production, whereas the charged substrates reduced production of both types, indicating a coupling of exogenous ROS generation and intracellular ROS production. Furthermore, PEG‐mediated ROS induction caused nuclear translocation of glyceraldehyde‐3‐phosphate dehydrogenase and an increase in caspase‐3 activity, confirming a link with apoptosis. PEG‐rich pro‐oxidant substrates caused cytoskeletal actin remodeling through β‐actin cleavage by caspase‐3 into fractins. The fractins co‐localized to the mitochondria and reduced the mitochondrial membrane potential. The remnant cytosolic β‐actin was polymerized and condensed, events consistent with apoptotic cell shrinkage. The cytoskeletal remodeling was integral to the further augmentation of intracellular ROS production. Conversely, the anti‐oxidant DT‐containing charged substrates suppressed the entire cascade of apoptotic progression. We demonstrate that ROS activity serves an important role in “outside‐in” signaling for cells grown on substrates: the ROS activity couples exogenous stress, driven by substrate composition, to changes in intracellular signaling. This signaling causes cell apoptosis, which is mediated by actin remodeling. J. Cell. Physiol. 218: 549–557, 2009. © 2008 Wiley‐Liss, Inc.     

Swainsonine Induces Caprine Luteal Cells Apoptosis via Mitochondrial‐Mediated Caspase‐Dependent Pathway

Swainsonine (SW) is an indolizidine alkaloid isolated from a number of poisonous plants. We have previously reported that SW inhibited luteal cell progesterone production by inducing caprine luteal cell apoptosis in vitro; however, the molecular mechanism of this phenomenon remains unclear. In this study, SW‐treated luteal cells showed apoptosis characteristics, including nuclear fragmentation, DNA ladder formation, and phosphatidylserine externalization. Further studies showed that SW activated caspase‐9 and caspase‐3, which subsequently cleaved poly(ADP‐ribose) polymerase. SW also increased in Bax/BcL‐2 ratios, promoted Bax translocation from the cytosol to mitochondria, and triggered the release of cytochrome c from mitochondria into the cytoplasm. However, Fas and Fas ligand induction or caspase‐8 activity did not appear any significant changes. Additional analysis also showed that pan‐caspase inhibitor, caspase‐9 inhibitor, or caspase‐3 inhibitor almost completely protected the cells from SW‐induced apoptosis, but not caspase‐8 inhibitor. Overall, these data demonstrated that SW induced luteal cells apoptosis through a mitochondrial‐mediated caspase‐dependent pathway.