Paclitaxel inhibits osteoclast formation and bone resorption via influencing mitotic cell cycle arrest and RANKL-induced activation of NF-κB and ERK

Pathological bone destruction (osteolysis) is a hallmark of many bone diseases including tumor metastasis to bone, locally osteolytic giant cell tumor (GCT) of bone, and Paget's disease. Paclitaxel is frequently prescribed in the treatment of several malignant tumors where it has been shown to exert beneficial effects on bone lesions. However, the mechanism(s) through which paclitaxel regulates osteoclast formation and function remain ill defined. In the present study, we demonstrate that paclitaxel dose-dependently inhibits receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in both RAW264.7 cells and mouse bone marrow macrophage (BMM) systems. In addition, paclitaxel treatment reduces the bone resorptive activity of human osteoclasts derived from GCT of bone, and attenuates lipopolysaccharide (LPS)-induced osteolysis in a mouse calvarial model. Complementary cellular and biochemical analyses revealed that paclitaxel induces mitotic arrest of osteoclastic precursor cells. Furthermore, luciferase reporter gene assays and western blot analysis indicate that paclitaxel modulates key RANKL-induced activation pathways that are essential to osteoclast formation including NF-κB and ERK. Collectively, our findings demonstrate a role for paclitaxel in the regulation of osteoclast formation and function and uncover potential mechanism(s) through which paclitaxel alleviates pathological osteolysis.     

Zinc induces ERK-dependent cell death through a specific Ras isoform

The effect of Zn on p53-independent cell death was examined in IIC9 embryonic fibroblasts. Despite the fact that these cells are p53-minus, Zn-mediated death occurs via an apoptotic mechanism. Death is facilitated by the presence of the Zn ionophore, pyrithione, indicating that intracellular Zn initiates the death response. Our investigations of the mechanism of Zn action demonstrate that Zn induces the death of IIC9 cells in a manner that is ERK-dependent. Expression of dn-(dominant negative)Ras attenuates ERK1/2 activation by Zn, and correspondingly reduces its cytotoxic effects. Raf-RBD pull-down experiments confirm that Zn treatment activates Ras and identified H-Ras as the specific isoform activated. This contrasts the activation of N-Ras that occurs when IIC9 cells are stimulated with thrombin. Thus, although the prolonged activation of the Ras/ERK pathway by Zn is similar to that seen when induced by mitogen, the distinguishing feature appears to be the isoform specificity of Ras activation. This work was supported by National Institute of Health Grants DK-52194 and AI-44458 (to J.A.C). P.L Blackwell was supported by an American Heart Association Grant in Aid 0555574Z (to C.K), K.J. Hughes was supported by the National Institute of Health Training Grant (GM008306) and Kimberly Creach was supported by St. Louis University Medical School Summer Research Fellowship.     

Human periodontal ligament fibroblasts stimulate osteoclastogenesis in response to compression force through TNF-α-mediated activation of CD4+ T cells

Periodontal ligament fibroblasts (PLF) sense and respond to mechanical stimuli and participate in alveolar bone resorption during orthodontic treatments. This study examined how PLF influence osteoclastogenesis from bone marrow-derived macrophages (BMM) after application of tension or compression force. We also investigated whether lymphocytes could be a primary stimulator of osteoclastic activation during alveolar bone remodeling. We found that mechanical forces inhibited osteoclastic differentiation from BMM in co-cultures with PLF, with PLF producing predominantly osteoprotegerin (OPG) rather than receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL). In particular, PLF increased the expression of tumor necrosis factor (TNF)-α in response to compression. Additional experiments showed the presence of CD4- and B220-positive cells with a subsequent increase in tartrate-resistant acid phosphatase (TRAP)-positive cells and RANKL expression only at the compression side of the force-subjected periodontal tissues. Exogenous TNF-α increased the number of TRAP-positive cells and pit formation in the co-cultures of BMM with Jurkat, but not with BJAB cells and this effect was almost completely inhibited by antibodies to TNF-α or TNF receptor. Collectively, the current findings suggest that PLF secrete relatively higher levels of TNF-α at the compression side than at the tension side and this imbalance leads to RANKL expression by activating CD4+ T cells, thereby facilitating bone resorption during orthodontic tooth movement.     

Plexin‐B1 is a GTPase activating protein for M‐Ras,remodelling dendrite morphology

Plexins are receptors for axonal guidance molecules known as semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin‐B1, induces axonal growth cone collapse by functioning as an R‐Ras GTPase activating protein (GAP). Here, we report that Plexin‐B1 shows GAP activity for M‐Ras, another member of the Ras family of GTPases. In cortical neurons, the expression of M‐Ras was upregulated during dendritic development. Knockdown of endogenous M‐Ras—but not R‐Ras—reduced dendritic outgrowth and branching, whereas overexpression of constitutively active M‐Ras, M‐Ras(Q71L), enhanced dendritic outgrowth and branching. Sema4D suppressed M‐Ras activity and reduced dendritic outgrowth and branching, but this reduction was blocked by M‐Ras(Q71L). M‐Ras(Q71L) stimulated extracellular signal‐regulated kinase (ERK) activation, inducing dendrite growth, whereas Sema4D suppressed ERK activity and down‐regulation of ERK was required for a Sema4D‐induced reduction of dendrite growth. Thus, we conclude that Plexin‐B1 is a dual functional GAP for R‐Ras and M‐Ras, remodelling axon and dendrite morphology, respectively.     

Involvement of Ras in extraembryonic endoderm differentiation of embryonic stem cells

Embryonic stem (ES) cells, derived from the inner cell mass of blastocyst can differentiate into multiple cell lineages. In this study, we examined the possible involvement of Ras in ES cell differentiation. We found that Ras was activated upon formation of embryoid bodies (EBs), an initial step in ES cell differentiation. When expressed during EB differentiation, a dominant-negative mutant of Ras suppressed induction of marker genes for extraembryonic endoderm differentiation, including GATA-4, GATA-6, alpha-fetoprotein, and hepatocyte nuclear factor 3beta, while an activated mutant promoted their induction. Expression of a Ras mutant that selectively activates the Raf/MEK/Erk pathway also enhanced induction of extraembryonic endoderm markers, and treatment with a MEK inhibitor resulted in their decreased expression. In addition, Ras stimulated downregulation of Nanog, a suppressor of endoderm differentiation in ES cells. These data suggest that Ras activation during EB differentiation plays a crucial role in initiation of extraembryonic endoderm differentiation.     

“RAF” neighborhood: Protein–protein interaction in the Raf/Mek/Erk pathway

The Raf/Mek/Erk signaling pathway, activated downstream of Ras primarily to promote proliferation, represents the best studied of the evolutionary conserved MAPK cascades. The investigation of the pathway has continued unabated since its discovery roughly 30 years ago. In the last decade, however, the identification of unexpected in vivo functions of pathway components, as well as the discovery of Raf mutations in human cancer, the ensuing quest for inhibitors, and the efforts to understand their mechanism of action, have boosted interest tremendously. From this large body of work, protein–protein interaction has emerged as a recurrent, crucial theme. This review focuses on the role of protein complexes in the regulation of the Raf/Mek/Erk pathway and in its cross-talk with other signaling cascades. Mapping these interactions and finding a way of exploiting them for therapeutic purposes is one of the challenges of future molecule-targeted therapy.     

Ras signals principally via Erk in G1 but cooperates with PI3K/Akt for Cyclin D induction and S-phase entry

Numerous studies exploring oncogenic Ras or manipulating physiological Ras signalling have established an irrefutable role for Ras as driver of cell cycle progression. Despite this wealth of information the precise signalling timeline and effectors engaged by Ras, particularly during G1, remain obscure as approaches for Ras inhibition are slow-acting and ill-suited for charting discrete Ras signalling episodes along the cell cycle. We have developed an approach based on the inducible recruitment of a Ras-GAP that enforces endogenous Ras inhibition within minutes. Applying this strategy to inhibit Ras stepwise in synchronous cell populations revealed that Ras signaling was required well into G1 for Cyclin D induction, pocket protein phosphorylation and S-phase entry, irrespective of whether cells emerged from quiescence or G2/M. Unexpectedly, Erk, and not PI3K/Akt or Ral was activated by Ras at mid-G1, albeit PI3K/Akt signalling was a necessary companion of Ras/Erk for sustaining cyclin-D levels and G1/S transition. Our findings chart mitogenic signaling by endogenous Ras during G1 and identify limited effector engagement restricted to Raf/MEK/Erk as a cogent distinction from oncogenic Ras signalling.     

Molecular diversity of CTX prophage in Vibrio cholerae

Aims: The objective of this study was to investigate the molecular diversity of CTX genetic element within toxigenic Vibrio cholerae genomes and to determine the genetic diversity of V. cholerae population collected in a 6‐year period (2004–2009) in Iran. Methods and Results: The results of mismatch amplification mutation assay (MAMA)‐PCR and sequencing showed cytosine nucleotide in positions 203 and 115 in all 50 El Tor V. cholerae strains, which is the same as classical ctxB sequence. One strain yielded amplicons with both El Tor and classical biotype primers in MAMA‐PCR indicative of presence of two copies of CTX phages with different genotypes (rstR^ET ctxB^class and rstR^ET ctxB^ET) integrated within the genome of this isolate, which suggested the integration of two different CTX phages at different occasions or point mutation in one copy of CTX. Sequencing and PCR analysis indicated the presence of hybrid CTX genotype (rstR^ET ctx^class) in 70·6% of the isolates; however, only El Tor RS1 phage has been integrated in flanking to the CTX phages with different genotypes. Conclusions: Enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR) and ribosomal gene spacer‐PCR (RS‐PCR) showed a relatively homogenous population in different years. Our findings indicate that sequence analysis of RS and ctxB regions has more discriminative power than restriction‐based methods. Significance and Impact of the Study: Investigating the molecular diversity of CTX prophage among V. cholerae strains helps to establish a new valuable database of genetic information about isolates, which is of great importance for epidemiologic studies in Iran and other countries encountering cholera epidemics.