PDZK1 Is a Novel Factor in Breast Cancer That Is Indirectly Regulated by Estrogen through IGF-1R and Promotes Estrogen-Mediated Growth

Although a relationship between PDZK1 expression and estrogen receptor (ER)-α stimulation has been suggested, the nature of such a connection and the function of PDZK1 in breast cancer remain unknown. Human tissue microarrays (cancer tissue: 262 cores; normal tissue: 87 cores) and breast cancer cell lines were used to conduct the study. We show that PDZK1 protein expression is tightly correlated with human breast malignancy, is negatively correlated with age and had no significant correlation with ER-α expression levels. PDZK1 exhibited an exclusive epithelial expression with mostly cytosolic subcellular localization. Additionally, 17β-estradiol induced PDZK1 expression above its basal level more than 24 h after treatment in MCF-7 cells. PDZK1 expression was indirectly regulated by ER-α stimulation, requiring insulinlike growth factor 1 receptor (IGF-1R) expression and function. The molecular link between PDZK1 and IGF-1R was supported by a significant correlation between protein and mRNA levels (r = 0.591, p < 0.001, and r = 0.537, p < 0.001, respectively) of the two factors in two different cohorts of human breast cancer tissues. Interestingly, PDZK1 knockdown in MCF-7 cells blocked ER-dependent growth and reduced c-Myc expression, whereas ectopic expression of PDZK1 enhanced cell proliferation in the presence or absence of 17β-estradiol potentially through an increase in c-Myc expression, suggesting that PDZK1 has oncogenic activity. PDKZ1 also appeared to interact with the Src/ER-α/epidermal growth factor receptor (EGFR) complex, but not with IGF-1R and enhanced EGFR-stimulated MEK/ERK1/2 signaling. Collectively, our results clarify the relationship between ER-α and PDZK1, propose a direct relationship between PDZK1 and IGF-1R, and identify a novel oncogenic activity for PDZK1 in breast cancer.     

Aldose Reductase Inhibition Prevents Allergic Airway Remodeling through PI3K/AKT/GSK3β Pathway in Mice

Background

Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR), an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA)-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs) and mouse lung fibroblasts (mLFs).

Methods

Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR) in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT) in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s) of airway remodeling.

Results

In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3.

Conclusion

Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.     

Anticarcinogenic effects of hexaamminecobalt(III) chloride in mice initiated with diethylnitrosamine

Hexaamminecobalt(III) chloride ([Co(NH₃)₆]Cl₃) was investigated for its antineoplastic role in relation to tumor marker enzymes, drug metabolizing enzymes, oxidative stress-related parameters, and histopathological analysis of liver and lung tissues of mice. Initiation was performed using a single intraperitoneal injection of diethylnitrosamine (DENA) at a carcinogenic dose of 90 mg/kg body weight. The cobalt complex supplementation at a dose of 100 ppm in drinking water was given ad libitum throughout the experimental period of 14 weeks. In comparison to lung, the cobalt complex supplementation was found to reverse DENA-induced biochemical changes more effectively in liver. Histological examination of liver and lung from DENA-initiated and cobalt-complex-supplemented mice showed considerable protection in the case of liver compared to that of lung. The involvement of the [Co(NH₃)₆]Cl₃ in modulating several factors associated with carcinogenesis induced by DENA thus showed its anticarcinogenic potential against chemically induced hepatocarcinogenesis.     

Cordycepin blocks lung injury-associated inflammation and promotes BRCA1-deficient breast cancer cell killing by effectively inhibiting PARP

Cordycepin has been shown to interfere with a myriad of molecular processes from RNA elongation to kinase activity, and prevents numerous inflammatory processes in animal models. Here we show in a mouse model of LPS-induced acute lung injury that cordycepin prevents airway neutrophilia via a robust blockade of expression of several inflammatory genes, including the adhesion molecule ICAM-1 and VCAM-1, the cytokine/chemokine MCP-1, MIP-1α, MIP-2 and KC, and the chemokine receptor CXCR2. Such a blockade appears to be related to a severe reduction in TNF-α expression. Interestingly, in an in vitro system of A549 epithelial cell inflammation, cordycepin effectively blocked LPS-induced, but not TNF-α-induced, VCAM-1 expression. Such effects correlated with a marked reduction in p65-NF-κB activation as assessed by its phosphorylation at serine-536 but without an apparent effect on its nuclear translocation. The effects of cordycepin on the expression of VCAM-1 and ICAM-1, and of NF-κB activation and nuclear translocation upon TNF-α stimulation resembled the effects achieved upon poly(ADP-ribose) polymerase (PARP) inhibition, suggesting that cordycepin may function as a PARP inhibitor. Indeed, cordycepin blocked H(2)O(2)-induced PARP activation in A549 cells. In a cell-free system, cordycepin inhibited PARP-1 activity at nanomolar concentrations. Similar to PARP inhibitors, cordycepin significantly induced killing of breast cancer susceptibility gene (BRCA1)-deficient MCF-7 cells, supporting its therapeutic use for the treatment of BRCA-deficient breast cancers. With added antiinflammatory characteristics, therapies that include cordycepin may prevent potential inflammation triggered by traditional chemotherapeutic drugs. Cordycepin, to the best of our knowledge, represents the first natural product possessing PARP inhibitory traits.     

Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment

The DNA binding activity of NF-κB is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-κB activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF. The phosphorylation and subsequent degradation of I-κBα as well as the serine 536 phosphorylation and nuclear translocation of p65 NF-κB were insufficient for VCAM-1 expression in response to TNF. The requirement for p50 NF-κB in TNF-induced VCAM-1 expression may be associated with its interaction with and phosphorylation by DNA-PK, which appears to be dominant over the requirement for p65 NF-κB activation. p50 NF-κB binding to its consensus sequence increased its susceptibility to phosphorylation by DNA-PK. Additionally, DNA-PK activity appeared to increase the association between p50/p50 and p50/p65 NF-κB dimers upon binding to DNA and after binding of p50 NF-κB to the VCAM-1 promoter. Analyses of the p50 NF-κB protein sequence revealed that both serine 20 and serine 227 at the amino terminus of the protein are putative sites for phosphorylation by DNA-PK. Mutation of serine 20 completely eliminated phosphorylation of p50 NF-κB by DNA-PK, suggesting that serine 20 is the only site in p50 NF-κB for phosphorylation by DNA-PK. Re-establishing wild-type p50 NF-κB, but not its serine 20/alanine mutant, in p50 NF-κB(-/-) fibroblasts reversed VCAM-1 expression after TNF treatment, demonstrating the importance of the serine 20 phosphorylation site in the induction of VCAM-1 expression. Together, these results elucidate a novel mechanism for the involvement of DNA-PK in the positive regulation of p50 NF-κB to drive VCAM-1 expression.     

Collapsin Response Mediator Protein 5 (CRMP5) Induces Mitophagy,Thereby Regulating Mitochondrion Numbers in Dendrites

Degradation of damaged mitochondria by mitophagy is an essential process to ensure cell homeostasis. Because neurons, which have a high energy demand, are particularly dependent on the mitochondrial dynamics, mitophagy represents a key mechanism to ensure correct neuronal function. Collapsin response mediator proteins 5 (CRMP5) belongs to a family of cytosolic proteins involved in axon guidance and neurite outgrowth signaling during neural development. CRMP5, which is highly expressed during brain development, plays an important role in the regulation of neuronal polarity by inhibiting dendrite outgrowth at early developmental stages. Here, we demonstrated that CRMP5 was present in vivo in brain mitochondria and is targeted to the inner mitochondrial membrane. The mitochondrial localization of CRMP5 induced mitophagy. CRMP5 overexpression triggered a drastic change in mitochondrial morphology, increased the number of lysosomes and double membrane vesicles termed autophagosomes, and enhanced the occurrence of microtubule-associated protein 1 light chain 3 (LC3) at the mitochondrial level. Moreover, the lipidated form of LC3, LC3-II, which triggers autophagy by insertion into autophagosomes, enhanced mitophagy initiation. Lysosomal marker translocates at the mitochondrial level, suggesting autophagosome-lysosome fusion, and induced the reduction of mitochondrial content via lysosomal degradation. We show that during early developmental stages the strong expression of endogenous CRMP5, which inhibits dendrite growth, correlated with a decrease of mitochondrial content. In contrast, the knockdown or a decrease of CRMP5 expression at later stages enhanced mitochondrion numbers in cultured neurons, suggesting that CRMP5 modulated these numbers. Our study elucidates a novel regulatory mechanism that utilizes CRMP5-induced mitophagy to orchestrate proper dendrite outgrowth and neuronal function.     

A novel peptide derived from vascular endothelial growth factor prevents amyloid beta aggregation and toxicity

Amyloid-β oligomers (Aβo) are the most pathologically relevant Aβ species in Alzheimer's disease (AD), because they induce early synaptic dysfunction that leads to learning and memory impairments. In contrast, increasing VEGF (Vascular Endothelial Growth Factor) brain levels have been shown to improve learning and memory processes, and to alleviate Aβ-mediated synapse dysfunction. Here, we designed a new peptide, the blocking peptide (BP), which is derived from an Aβo-targeted domain of the VEGF protein, and investigated its effect on Aβ-associated toxicity. Using a combination of biochemical, 3D and ultrastructural imaging, and electrophysiological approaches, we demonstrated that BP strongly interacts with Aβo and blocks Aβ fibrillar aggregation process, leading to the formation of Aβ amorphous aggregates. BP further impedes the formation of structured Aβo and prevents their pathogenic binding to synapses. Importantly, acute BP treatment successfully rescues long-term potentiation (LTP) in the APP/PS1 mouse model of AD, at an age when LTP is highly impaired in hippocampal slices. Moreover, BP is also able to block the interaction between Aβo and VEGF, which suggests a dual mechanism aimed at both trapping Aβo and releasing VEGF to alleviate Aβo-induced synaptic damage. Our findings provide evidence for a neutralizing effect of the BP on Aβ aggregation process and pathogenic action, highlighting a potential new therapeutic strategy.     

Correlation between PDZK1, Cdc37, Akt and Breast Cancer Malignancy: The Role of PDZK1 in Cell Growth through Akt Stabilization by Increasing and Interacting with Cdc37

PDZ domain containing 1 (PDZK1) is a scaffold protein that plays a role in the fate of several proteins. Estrogen can induce PDZK1 gene expression; however, our recent report showed that PDZK1 expression in the breast cancer cell line MCF-7 is indirect and involves insulin-like growth factor (IGF)-1 receptor function. Such a relationship was established in cell culture systems and human breast cancer tissues. Here we show that overexpression of PDZK1 promoted an increase in cyclin D1 and enhanced anchorage-independent growth of MCF-7 cells in the absence of 17β-estradiol, suggesting that PDZK1 harbors oncogenic activity. Indeed, PDKZ1 overexpression enhanced epidermal growth factor receptor (EGFR)-stimulated MEK/ERK1/2 signaling and IGF-induced Akt phosphorylation. PDZK1 appeared to play this role, in part, by stabilizing the integrity of the growth promoting factors Akt, human epidermal growth factor receptor 2 (Her2/Neu) and EGFR. Increased Akt levels occurred via a decrease in the ubiquitination of the kinase. PDZK1 overexpression was associated with resistance to paclitaxel/5-fluorouracil/etoposide only at low concentrations. Although the increased stability of Akt was sensitive to heat shock protein 90 (HSP90) inhibition, increased levels of the cochaperone cell division cycle 37 (Cdc37), as well as its ability to bind PDZK1, appear to play a larger role in kinase stability. Using human tissue microarrays, we show strong positive correlation between PDZK1, Akt and Cdc37 protein levels, and all correlated with human breast malignancy. There were no positive correlations between PDZK1 and Cdc37 at the mRNA levels, confirming our in vitro studies. These results demonstrate a relationship between PDZK1, Akt and Cdc37, and potentially Her2/Neu and EGFR, in breast cancer, representing a new axis that can be targeted therapeutically to reduce the burden of human breast cancer.     

Nuclear translocation of p65 NF-kappaB is sufficient for VCAM-1, but not ICAM-1, expression in TNF-stimulated smooth muscle cells: Differential requirement for PARP-1 expression and interaction

Although nuclear translocation of NF-kappaB and subsequent binding to promoters of ICAM-1 and VCAM-1 have been shown to be decisive for their expression, a number of discrepancies in the expression patterns of these adhesion molecules have been reported in both cell culture systems and disease settings, including atherosclerosis, asthma, and autoimmune diseases. Here we show that while p65 NF-kappaB nuclear translocation in TNF-treated smooth muscle cells (SMCs) was sufficient for the expression of VCAM-1, expression of ICAM-1 showed a critical requirement for PARP-1. I-kappaBalpha phosphorylation and subsequent degradation were virtually identical in both TNF-treated wild-type and PARP-1-/- SMCs. VCAM-1 expression in TNF-treated PARP-1-/- SMCs was completely inhibited by the NF-kappaB inhibitor, pyrrolidine dithiocarbamate, confirming that VCAM-1 expression was indeed NF-kappaB-dependent. The expression of both VCAM-1 and ICAM-1 was associated with a transient interaction between PARP-1 and p65 NF-kappaB when examined in the fibroblastic cell line, COS-7, and in the airway epithelial cell line, A549. Such interactions were confirmed using florescence resonance energy transfer analysis. Protein acetylation activity, mediated by p300/CBP, was required for both VCAM-1 and ICAM-1 expression in TNF-treated SMCs; however, the interaction of PARP-1 with p300/CBP was dispensable for VCAM-1 expression. These findings indicate that p65 NF-kappaB nuclear translocation may be sufficient for certain genes (e.g., VCAM-1) while insufficient for others (e.g., ICAM-1), thus providing a novel insight into the role of NF-kappaB in driving target gene expression. Furthermore, the data suggest a differential requirement for PARP-1 expression in inflammatory processes.     

Studies on mode of action of hexaammine co(III) chloride against diethylnitrosamine‐induced hepatocarcinogenesis in mice

We recently reported the anticarcinogenic potential of hexaammine cobalt(III) chloride, a synthetic complex of cobalt, on diethylnitrosamine (DENA)‐induced carcinogenesis. The present study was conducted to ascertain the possible mode of action of this compound on DENA‐induced hepatocarcinogenesis in male BALB/c mice. Time course evaluation of liver injury markers showed that the low dose of the compound is more effective in ameliorating DENA‐induced changes when administered for longer duration of time. Long‐term exposure of the compound significantly reversed the levels of diacylgylcerol (DAG) and nitric oxide synthase (NOS) induced by DENA, thus suggesting that the compound may hinder the process of chemical carcinogenesis potentially by downregulating the signal transduction mechanism involving DAG and NOS. Furthermore, short‐term intraperitoneal injection of the compound to mice 26 weeks after DENA initiation reduced the cell viability count in preneoplstic liver lesions in a dose‐dependent manner. In conclusion, our results showed that anticarcinogenic effects of hexaammine cobalt(III) chloride result from its influence on signal transduction events mediated through DAG together with its direct cytotoxic action against preneoplastic hepatic lesions induced by DENA in mice. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:193–201, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20280