Retraction: Extracellular regulated kinase 5 mediates osteoporosis through modulating viability and apoptosis of osteoblasts in ovariectomized rats.

Postmenopausal osteoporosis is a common condition characterized by the increase and activation of osteoclasts. The present study aimed to investigate the effects of extracellular signal-regulated kinase (ERK) 5 (ERK-5) on postmenopausal osteoporosis by regulating the biological behaviors of osteoblasts. Sprague–Dawley (SD) rats were ovariectomized to develop an osteoporosis model. A lentivirus packaging system was employed to generate lentiviruses capable of up- or down-regulating the expression of ERK-5 in ovariectomized rats. The femoral biomechanical properties, bone mineral density (BMD), contents of calcium (Ca), phosphorus (P) and alkaline phosphatase (ALP) and bone turnover markers in rats, as well as viability, cycle and apoptosis of osteoblasts and ALP activity in osteoblasts were measured in the ovariectomized rats so as to explore the functional significance of ERK-5 in postmenopausal osteoporosis. The femoral mechanical strength of ovariectomized rats was enhanced by overexpression of ERK-5. Meanwhile femoral BMD, and bone metabolism were increased, and bone turnover normalized in the ovariectomized rats when ERK-5 was overexpressed. Lentivirus-mediated ERK-5 overexpression in osteoblasts was observed to inhibit osteoblast apoptosis, and promote viability, accompanied with increased ALP activity. Taken together, ERK-5 could decelerate osteoblast apoptosis and improve postmenopausal osteoporosis by increasing osteoblast viability. Thus, our study provides further understanding on a promising therapeutic target for postmenopausal osteoporosis.

Bioscience Reports has been made aware of potential issues surrounding the scientific validity of this paper and hence is issuing an Expression of Concern to notify readers whilst the Editorial Office investigates. Irregularities have been noted in the flow cytometry graphs – the authors were contacted for comment but have not yet responded or provided requested raw data.

Expression of Concern: Extracellular regulated kinase 5 mediates osteoporosis through modulating viability and apoptosis of osteoblasts in ovariectomized rats.

Cytosolic phospholipase A(2) (cPLA(2)) is activated by phosphorylation at serine-505 (S505) by extracellular regulated kinase 1/2 (ERK1/2). However, rat brain calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates recombinant cPLA(2) at serine-515 (S515) and increases its activity in vitro. We have studied the sites of cPLA(2) phosphorylation and their significance in arachidonic acid (AA) release in response to norepinephrine (NE) in vivo in rabbit vascular smooth muscle cells (VSMCs) using specific anti-phospho-S515- and -S505 cPLA(2) antibodies and by mutagenesis of S515 and S505 to alanine. NE increased the phosphorylation of cPLA(2) at S515, followed by phosphorylation of ERK1/2 and consequently phosphorylation of cPLA(2) at S505. The CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine attenuated cPLA(2) at S515 and S505, whereas the ERK1/2 inhibitor U0126 reduced phosphorylation at S505 but not at S515. NE in cells transduced with adenovirus carrying enhanced cyan fluorescent protein cPLA(2) wild type caused phosphorylation at S515 and S505 and increased AA release. Expression of the S515A mutant in VSMCs reduced the phosphorylation of S505, ERK1/2, and AA release in response to NE. Transduction with a double mutant (S515A/S505A) blocked the phosphorylation of cPLA(2) and AA release. These data suggest that the NE-stimulated phosphorylation of cPLA(2) at S515 is required for the phosphorylation of S505 by ERK1/2 and that both sites of phosphorylation are important for AA release in VSMCs.

Inhibition of acetylcholine-induced activation of extracellular regulated protein kinase prevents the encoding of an inhibitory avoidance response in the rat

Hydroxyapatite nanoparticles (nano-HAP) have been reported to cause inflammatory reactions. Here, we aimed to compare the effects of four types of nano-HAP with different nanocrystal morphologies (short rod-like, long rod-like, spherical or needle-shaped crystals) and sizes (10-20, 10-30 or 20-40 nm) on growth inhibition and apoptosis in primary cultured rat osteoblasts. The osteoblasts was treated with the four types of nano-HAP at various concentrations (20, 40, 60, 80 or 100 mg l⁻¹). The nano-HAP specific surface area was detected using the Brunauer, Emmet and Teller method. The cell growth rate was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; apoptotic alterations and the level of reactive oxygen species in osteoblasts were measured using flow cytometry; and the amounts of apoptotic p53 and cytochrome c proteins were measured using western blotting. We observed that all four types of nano-HAP inhibited the growth of osteoblasts in a dose-dependent manner. These nano-HAP significantly induced apoptosis in osteoblasts. Nano-HAP with smaller specific surface areas induced lower apoptosis rates. The needle-shaped and the short rod-like particles induced greater cellular injury than the spherical and long rod-like particles, respectively. The increased apoptosis rates were accompanied by increased p53 and cytochrome c expression. These findings indicate that nano-HAP inhibit the activity of osteoblasts and also induce the apoptosis of osteoblasts in vitro. These findings also suggest that the nano-HAP-induced apoptotic pathway is mediated by a mitochondrial-dependent pathway. Moreover, the sizes, morphologies and concentrations of nano-HAP have significant effects on the apoptotic level.

CaMKⅡ mediates cadmium induced apoptosis in rat primary osteoblasts through MAPK activation and endoplasmic reticulum stress

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including cell survival. PI3Kγ, a member of the PI3K family, is activated in response to G-protein coupled receptor (GPCR) stimulation leading to extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, less is known about the underlying mechanisms of PI3Kγ-directed ERK activation. Knockdown of PI3Kγ showed that PI3Kγ not only regulates ERK phosphorylation in response to GPCR stimulation but also to receptor tyrosine kinase activation in HEK 293 cells. The key role of PI3Kγ in ERK activation was further validated by loss of insulin-stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs demonstrating a kinase-independent role of PI3Kγ in regulating ERK function. Mechanistic studies showed that PI3Kγ regulates ERK activation by inhibiting ERK dephosphorylation following stimulation thereby, sustaining ERK phosphorylation and activation. Critically, PI3Kγ regulates ERK dephosphorylating phosphatase PP2A by interacting and sequestering PP2A from ERK maintaining ERK phosphorylation, which is evidenced by increased PP2A association with ERK in KO MEFs. Consistently, ERK activation was completely abolished in KO MEFs following carvedilol or insulin suggesting an essential role for PI3Kγ in ERK activation pathway. Correspondingly, primary cardiac fibroblasts isolated from KO mice showed complete loss of insulin-stimulated ERK phosphorylation compared to WT mice. This is intriguing given that GSK3 phosphorylation and not ERK phosphorylation is regulated by inhibition of PP2A through kinase-independent mechanism of PI3Kγ in the total cardiac lysates. Even though GSK3 and ERK are substrates for PP2A, our findings that ERK is regulated by kinase-independent function PI3Kγ suggest the existence of this unique regulation in fibroblasts and not in cardiomyocytes. Thus, kinase activity of PI3Kγ may contribute to cardiac-pathology while kinase-independent function could be beneficial and will be discussed in presentation.

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including survival. PI3Kγ, a member of the PI3K family activated by G-protein coupled receptor (GPCR), is known to be a critical player in activation of extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, the exact mechanism by which PI3Kγ plays a role in ERK activation is not clearly understood. Our studies show that PI3Kγ plays a crucial role in enhancing the tone of ERK activation as use of PI3K inhibitors reduced GPCR stimulated ERK phosphorylation in HEK293 cells. siRNA knockdown of PI3Kγ resulted in loss of ERK phosphorylation through GPCRs (β-adrenergic) as well as receptor tyrosine kinases. The role of PI3Kγ in ERK activation was further corroborated by loss of insulin stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs suggesting a kinase-independent role of PI3Kγ in regulating ERK function. Indepth mechanistic studies showed that PI3Kγ mediated activation of ERK by inhibiting ERK dephosphorylation following stimulation, thus stabilizing the ERK phosphorylation. PI3Kγ physically disrupts the interaction between ERK and ERK dephosphorylating phosphatase PP2A as evidenced by increase in phosphatase association with ERK in KO MEFs. Consistent with this observation, ERK activation was completely abolished in KO MEFs following carvedilol suggesting an essential role for PI3Kγ in cardio-protective ERK activation pathway. In this context, it is known that transverse aortic constriction (TAC) in mice leads to increase in ERK activation in the hearts and is also associated with concurrent up-regulation of PI3Kγ suggesting a key role for kinase-independent function of PI3Kγ in activating and maintaining the ERK signaling cascade. These indepth cellular studies and observation from our TAC studies led us to believe that kinase-dependent function of PI3Kγ may contribute to pathology while kinase-independent function may be cardio-protective through inhibition of PP2A by PI3Kγ. This novel signaling mechanism by PI3Kγ will be presented.

Impact of Redox Modifications on ERK2 Substrate Phosphorylation

Axin 1 knockdown inhibits osteoblastic apoptosis induced by Porphyromonas gingivalis lipopolysaccharide

A consistent pattern of response has been observed when FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3- internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3-ITD AML blasts co-cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti-apoptotic effect is mediated through a combination of direct cell-cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine-activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal-mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3-ITD AML.

Long noncoding RNAs (lncRNAs) have been revealed to be related to multiple physiological and pathology processes such as development, carcinogenesis, and osteogenesis. It is reported that lncRNAs might exert function in osteoblast differentiation and bone formation. Here, we determined this study to clarify whether lncRNA CCAT1 could regulate osteoblast proliferation and differentiation in ovariectomized rats with osteoporosis. The osteoporosis models were established by bilateral ovariectomy and treated with CCAT1 siRNAs to discuss the effect of CCAT1 on pathological changes and osteocyte apoptosis in ovariectomized rats with osteoporosis. The osteoblasts from ovariectomized rats were cultured in vitro, which were then treated with CCAT1 siRNAs to explore the role of CCAT1 in osteoblast proliferation and differentiation. Moreover, the relationships among CCAT1, miR-34a-5p, and SMURF2 were confirmed. CCAT1 and SMURF2 were amplified while miR-34a-5p expression was inhibited in bone tissues and osteoblasts of ovariectomized rats with osteoporosis. Inhibited CCAT1 improved pathology and restricted osteocyte apoptosis of bone tissues in ovariectomized rats with osteoporosis in vivo, and also enhanced differentiation, mineralization abilities, and proliferation, and suppressed apoptosis of osteoblasts from ovariectomized rats in vitro through upregulating miR-34a-5p expression. LncRNA CCAT1 could competitively bind with miR-34a-5p to prevent the degradation of its target gene SMURF2. Results of this research suggested that the CCAT1 inhibits the proliferation and differentiation of osteoblasts in rats with osteoporosis by binding to miR-34a-5p, providing novel biomarkers for osteoporosis treatment.

Phosphoinositide 3 Kinase γ (PI3Kγ) regulates anti-apoptotic Akt signaling. Previous studies have established a role for PI3Kγ in cardiac fibrosis, a key underlying cause of heart failure. However, less is known about the mechanism by which PI3Kγ regulates cardiac myofibroblast differentiation, hallmark of tissue fibrosis, characterized by smooth muscle α-actin (αSMA) overexpression. Measurement of αSMA abundance in cardiac lysates from PI3Kγ null mice (PI3Kγ -/- ) showed significant baseline and pressure overload induced upregulation compared to wildtype (WT), indicating that loss of PI3Kγ predisposes the hearts towards fibrosis. Furthermore, isolated cardiac fibroblasts (CF) from PI3Kγ -/- exhibited a myofibroblast phenotype with αSMA in stress fibers. Correspondingly, immunoblotting showed significantly higher αSMA in PI3Kγ -/- CF than WT. It has been previously shown that fibroblast growth factor mediated activation of the signaling pathway downregulates αSMA and that this inhibition of αSMA expression is through negative regulation by extracellular regulated kinase (ERK). To understand whether PI3Kγ regulates ERK signaling in a cell specific manner and thereby possibly αSMA, the phosphorylation of ERK by insulin stimulation were compared in CF isolated from WT and PI3Kγ -/- . Intriguingly, there was significant loss of ERK phosphorylation in CF from PI3Kγ -/- when compared to CF from WT. However, ERK phosphorylation was not altered in cardiomyocytes (CM) due to the absence of PI3Kγ. Confirming this differential regulation of ERK in CM and CF, there was no change in the association of ERK and protein phosphatase 2A (PP2A) in CM of WT and PI3Kγ -/- . However, there was increased association of PP2A with ERK in CF of PI3Kγ -/- when compared to CF from WT. This is because in the CM, ERK is regulated by Dual Specificity Phosphatase (DUSP8). However, in the CF, PP2A is the major regulator of ERK and thus PI3Kγ plays a major role in ERK signaling in CF. Consistent with previous observations, we found that association of DUSP8 with ERK was not observed in CF. Moreover, ERK-DUSP8 interaction was not dependent on the presence or absence of PI3Kγ. These data indicate that PI3Kγ regulates signaling pathways in a cell specific manner with respect to cardiac remodeling.

We recently characterized the association of the 44-kDa mitogen-activated protein kinase, also known as extracellular-regulated kinase 1 (ERK1), with the 90-kDa ribosomal S6 kinase (pp90rsk), one of its putative substrates in intact PC12 cells. Using antibodies to ERK1 that precipitate a functional ERK1.pp90rsk phosphoprotein complex, we demonstrate here the regulation of both kinases by various stimuli. In mouse fibroblasts expressing human insulin receptors, insulin and vanadate swiftly stimulated ERK1 activity within 5 min. While the hormonal effect was short-lived, vanadate led to a first peak followed by a progressively increasing second phase. In PC12 cells, epidermal growth factor, which is a growth promoting factor, provokes a rapid but evanescent activation of ERK1. In contrast, nerve growth factor (NGF), which acts as a neuronal differentiation factor for PC12 cells, induced a swift monophasic response followed by a sustained second phase. This strikingly different pattern of ERK1 stimulation by NGF and epidermal growth factor was associated to a contrasting effect on ERK1 cellular translocation. Thus, NGF induced a nuclear translocation of ERK1, while epidermal growth factor was without noticeable effect on ERK1 localization. In both cell systems all effectors tested stimulated ERK1 phosphorylation on both threonine and tyrosine residues in an 1:1 ratio. During ERK1 inactivation, phosphothreonine and phosphotyrosine were dephosphorylated in a similar fashion. Concurrent with ERK1 activation was the de novo appearance of phosphothreonine and an increase in phosphoserine on pp90rsk. The pp90rsk phosphothreonine content paralleled the ERK1 activity more closely than the phosphoserine level. These results provide compelling evidence that in fibroblasts and PC12 cells ERK1 plays a direct role in the phosphorylation of pp90rsk and that pp90rsk represents a physiologically relevant substrate of extracellular-regulated kinases. Finally, we would like to suggest that the differentiating action of NGF in PC12 cells might be due, at least in part, to the conjunction of its sustained and robust stimulation of ERK1 and pp90rsk, and of its induction of ERK1 nuclear translocation.