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Supplementary Figure Legends 1-2 from Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness

Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM. Cancer Res; 70(1); 418–27

Supplementary Table 1 from Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness

Supplementary Figure 2 from Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness

Supplementary Figure 1 from Receptor Channel TRPC6 Is a Key Mediator of Notch-Driven Glioblastoma Growth and Invasiveness

Parkinson's disease (PD) is a progressive motor disease of unknown etiology. Although neuroprotective ability of endogenous bile acid, tauroursodeoxycholic acid (TUDCA), shown in various diseases, including an acute model of PD,the potential therapeutic role of TUDCA in progressive models of PD that exhibit all aspects of PD has not been elucidated. In the present study, mice were assigned to one of four treatment groups: (1) Probenecid (PROB); (2) TUDCA, (3) MPTP + PROB (MPTPp); and (3) TUDCA + MPTPp.

The accumulation of amyloid-β (Aβ) is a characteristic event in the pathogenesis of Alzheimer’s disease (AD). Aquaporin 1 (AQP1) is a membrane water channel protein belonging to the AQP family. AQP1 levels are elevated in the cerebral cortex during the early stages of AD, but the role of AQP1 in AD pathogenesis is unclear. We first determined the expression and distribution of AQP1 in brain tissue samples of AD patients and two AD mouse models (3xTg-AD and 5xFAD). AQP1 accumulation was observed in vulnerable neurons in the cerebral cortex of AD patients, and in neurons affected by the Aβ or tau pathology in the 3xTg-AD and 5xFAD mice. AQP1 levels increased in neurons as aging progressed in the AD mouse models. Stress stimuli increased AQP1 in primary cortical neurons. In response to cellular stress, AQP1 appeared to translocate to endocytic compartments of β- and γ-secretase activities. Ectopic expression of AQP1 in human neuroblastoma cells overexpressing amyloid precussir protein (APP) with the Swedish mutations reduced β-secretase (BACE1)-mediated cleavage of APP and reduced Aβ production without altering the nonamyloidogenic pathway. Conversely, knockdown of AQP1 enhanced BACE1 activity and Aβ production. Immunoprecipitation experiments showed that AQP1 decreased the association of BACE1 with APP. Analysis of a human database showed that the amount of Aβ decreases as the expression of AQP1 increases. These results suggest that the upregulation of AQP1 is an adaptive response of neurons to stress that reduces Aβ production by inhibiting the binding between BACE1 and APP.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease, whose hallmark is the loss of dopamine terminals in the substantia nigra pars compacta (SNpc). PD is usually diagnosed after the appearance of motor symptoms, when about 70% of neurons in the SNpc have already been lost. Because of that, it is important to search for new methods that aid in the early diagnosis of PD. In recent years, microRNAs (miRs) have emerged as potential biomarkers for a variety of diseases and hold the potential to be used to aid in the diagnosis of PD. Therefore, the aim of this study was to characterize if specific miRs are differentially expressed in serum in a mouse model of PD. To induce PD-like damage, mice were subcutaneously injected with 25 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) by administering 10 doses over a period of 5 weeks, with 3.5 days between doses. Expression of 71 different microRNAs was quantified in serum separated from blood collected at day 35, using next-generation sequencing. Histological analysis and quantification of neurotransmitters were performed to confirm dopaminergic neurodegeneration. Chronic MPTP treatment induced loss of dopaminergic terminals in the SNpc and caudate putamen, confirmed by a decrease in the number of tyrosine hydroxylase and dopamine transporter positive cells. In addition, MPTP decreased the concentration of dopamine and its metabolites in the SNpc, simulating the damage observed in PD. From the 71 miRs analyzed, only 4 were differentially expressed after MPTP treatment. Serum levels of miR19b, miR124, miR126a and miR133b were significantly decreased in MPTP-treated mice compared to control. These data suggest that specific miRs are downregulated in a pre-clinical model of PD and hold the potential to be used as biomarkers to aid in the diagnosis of this disease. Further experiments need to be conducted to validate the use of these miRs as biomarkers of PD in additional pre-clinical models as well as in samples from patients diagnosed with PD.

Acute ischemic stroke causes a high rate of deaths and permanent neurological deficits in survivors. Current interventional treatment, in the form of enzymatic thrombolysis, benefits only a small percentage of patients. Brain ischemia triggers mobilization of innate immunity, specifically the complement system and Toll-like receptors (TLRs), ultimately leading to an exaggerated inflammatory response. Here we demonstrate that intravenous immunoglobulin (IVIG), a scavenger of potentially harmful complement fragments, and C1-esterase inhibitor (C1-INH), an inhibitor of complement activation, exert a beneficial effect on the outcome of experimental brain ischemia (I) and reperfusion (R) injury induced by transient occlusion of middle cerebral artery in mice. Both IVIG and C1-INH significantly and in a dose–responsive manner reduced brain infarction size, neurological deficit and mortality when administered to male mice 30 min before ischemia or up to 6 h after the onset of reperfusion. When combined, suboptimal doses of IVIG and C1-INH potentiated each other’s neuro-protective therapeutic effects. Complement C3 and TLR2 signals were colocalized and significantly greater in brain cells adjacent to infracted brain lesions when compared to the corresponding regions of the contralateral hemisphere and to control (sham) mice. Treatment with IVIG and C1-INH effectively reduced deposition of C3b and downregulated excessive TLR2 and p-JNK1 expression at the site of I/R injury. Taken together, these results provide a rationale for potential use of IVIG and C1-INH, alone or in combination with ischemic stroke and other neurological conditions that involve inappropriately activated components of the innate immune system.

Adipose tissue is a highly vascularized endocrine organ, and its secretion profiles may vary with obesity. Adiponectin is secreted by adipocytes that make up adipose tissue. Worldwide, obesity has been designated a serious health problem among women and is associated with a variety of metabolic disorders and an increased risk of developing cancer of the cervix, ovaries, uterus (uterine/endometrial), and breast. In this review, the potential link between obesity and female-specific malignancies is comprehensively presented by discussing significant features of the intriguing and complex molecule, adiponectin, with a focus on recent findings highlighting its molecular mechanism of action in female-specific carcinogenesis.

Objective Multiple sclerosis (MS) is a debilitating neurological disorder involving an autoimmune reaction to oligodendrocytes and degeneration of the axons they ensheath in the CNS. Because the damage to oligodendrocytes and axons involves local inflammation and associated oxidative stress, we tested the therapeutic efficacy of combined treatment with a potent anti-inflammatory thalidomide analog (lenalidomide) and novel synthetic anti-oxidant cerium oxide nanoparticles (nanoceria) in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Methods C57BL/6 mice were randomly assigned to a control (no EAE) group, or one of the four myelin oligodendrocyte glycoprotein-induced EAE groups: vehicle, lenalidomide, nanoceria, or lenalidomide plus nanoceria. During a 23 day period, clinical EAE symptoms were evaluated daily, and MRI brain scans were performed at 11–13 days and 20–22 days. Histological and biochemical analyses of brain tissue samples were performed to quantify myelin loss and local inflammation. Results Lenalidomide treatment alone delayed symptom onset, while nanoceria treatment had no effect on symptom onset or severity, but did promote recovery; lenalidomide and nanoceria each significantly attenuated white matter pathology and associated inflammation. Combined treatment with lenalidomide and nanoceria resulted in a near elimination of EAE symptoms, and reduced white matter pathology and inflammatory cell responses to a much greater extent than either treatment alone. Interpretation By suppressing inflammation and oxidative stress, combined treatment with lenalidomide and nanoceria can reduce demyelination and associated neurological symptoms in EAE mice. Our preclinical data suggest a potential application of this combination therapy in MS.

Tissue engineering and regenerative medicine aim to achieve functional restoration of tissue or cells damaged through disease, aging, or trauma. Advancement of tissue engineering requires innovation in the field of three-dimensional scaffolding and functionalization with bioactive molecules. Nanotechnology offers advanced materials with patterned nano-morphologies for cell growth and different molecular substrates that can support cell survival and functions. Cerium oxide nanoparticles (nanoceria) can control intracellular as well as extracellular reactive oxygen and nitrogen species. Recent findings suggest that nanoceria can enhance long-term cell survival, enable cell migration and proliferation, and promote stem cell differentiation. Moreover, the self-regenerative property of nanoceria permits a small dose to remain catalytically active for an extended time. This review summarizes the possibilities and applications of nanoceria in the field of tissue engineering and regenerative medicine.

Although selective neurodegeneration of nigro-striatal dopaminergic neurons is widely accepted as a cause of Parkinson's disease (PD), the role of vascular components in the brain in PD pathology is not well understood. However, the neurodegeneration seen in PD is known to be associated with neuroinflammatory-like changes that can affect or be associated with brain vascular function. Thus, dysfunction of the capillary endothelial cell component of neurovascular units present in the brain may contribute to the damage to dopaminergic neurons that occurs in PD. An animal model of PD employing acute, sub-acute and chronic exposures of mice to methyl-phenyl-tetrahydropyridine (MPTP) was used to determine the extent to which brain vasculature may be damaged in PD. Fluoro-Turquoise gelatin labeling of microvessels and endothelial cells was used to determine the extent of vascular damage produced by MPTP. In addition, tyrosine hydroxylase (TH) and NeuN were employed to detect and quantify dopaminergic neuron damage in the striatum (CPu) and substantia nigra (SNc). Gliosis was evaluated through GFAP immunohistochemistry. MPTP treatment drastically reduced TH immunoreactive neurons in the SNc (20.68 ± 2.83 in acute; 22.98 ± 2.14 in sub-acute; 10.20 ± 2.24 in chronic vs 34.88 ± 2.91 in controls; p0.001). Similarly, TH immunoreactive terminals were dramatically reduced in the CPu of MPTP treated mice. Additionally, all three MPTP exposures resulted in a decrease in the intensity, length, and number of vessels in both CPu and SNc. Degenerative vascular changes such as endothelial cell 'clusters' were also observed after MPTP suggesting that vasculature damage may be modifying the availability of nutrients and exposing blood cells and/or toxic substances to neurons and glia. In summary, vascular damage and degeneration could be an additional exacerbating factor in the progression of PD, and therapeutics that protect and insure vascular integrity may be novel treatments for PD.

Rapid and effective wound healing requires a coordinated cellular response involving fibroblasts, keratinocytes and vascular endothelial cells (VECs). Impaired wound healing can result in multiple adverse health outcomes and, although antibiotics can forestall infection, treatments that accelerate wound healing are lacking. We now report that topical application of water soluble cerium oxide nanoparticles (Nanoceria) accelerates the healing of full-thickness dermal wounds in mice by a mechanism that involves enhancement of the proliferation and migration of fibroblasts, keratinocytes and VECs. The Nanoceria penetrated into the wound tissue and reduced oxidative damage to cellular membranes and proteins, suggesting a therapeutic potential for topical treatment of wounds with antioxidant nanoparticles.

Tumor necrosis factor-α (TNF) plays a prominent role in the brain damage and functional deficits that result from ischemic stroke. It was recently reported that the thalidomide analog 3,6'-dithiothalidomide (3,6'-DT) can selectively inhibit the synthesis of TNF in cultured cells. We therefore tested the therapeutic potential of 3,6'-DT in a mouse model of focal ischemic stroke. Administration of 3,6'-DT immediately prior to a stroke or within 3 hr after the stroke reduced infarct volume, neuronal death, and neurological deficits, whereas thalidomide was effective only when administered prior to stroke. Neuroprotection was accompanied by decreased inflammation; 3,6'-DT-treated mice exhibited reduced expression of TNF, interleukin-1β, and inducible nitric oxide synthase; reduced numbers of activated microglia/macrophages, astrocytes, and neutrophils; and reduced expression of intercellular adhesion molecule-1 in the ischemic brain tissue. 3,6'-DT treatment attenuated stroke-induced disruption of the blood-brain barrier by a mechanism that appears to involve suppression of matrix metalloproteinase-9 and preservation of occludin. Treatment with 3,6'-DT did not reduce ischemic brain damage in mice lacking TNF receptors, consistent with a critical role for suppression of TNF production and TNF signaling in the therapeutic action of 3,6'-DT. These findings suggest that anti-inflammatory mechanisms underlie the therapeutic actions of 3,6-DT in an animal model of stroke.

Chronic intake of nicotine can impair hippocampal plasticity, but the underlying mechanism is poorly understood. Here, we demonstrate that chronic nicotine administration in adult rats inactivates the cyclic AMP-response element binding protein (CREB), a transcription factor that regulates neurogenesis and other plasticity-related processes necessary for learning and memory. Consequently, we showed that impaired CREB signaling is associated with a significant decline in the production of new neurons in the dentate gyrus. Combining retrovirus labeling with gene expression approaches, we found that chronic nicotine administration reduces the number of adult-generated granule neurons by decreasing the survival of newborn cells but not the proliferation of progenitor cells. Additionally, we found that retroviral-mediated expression of a constitutively active CREB in the dentate gyrus rescues survival of newborn cells and reverses the nicotine-induced decline in the number of mature granule neurons. Prolonged nicotine exposure also compromises CREB activation and reduces the viability of progenitor cells in vitro, thereby suggesting that nicotine may exert its adverse effects directly on immature cells in vivo. Taken together, these data demonstrate that inhibition of CREB activation is responsible for the nicotine-induced impairment of hippocampal plasticity.

Background and Purpose— Activation of Notch worsens ischemic brain damage as antisense knockdown or pharmacological inhibition of the Notch pathway reduces the infarct size and improves the functional outcome in a mouse model of stroke. We sought to determine whether Notch activation contributes to postischemic inflammation by directly modulating the microglial innate response. Methods— The microglial response and the attendant inflammatory reaction were evaluated in Notch1 antisense transgenic (Tg) and in nontransgenic (non-Tg) mice subjected to middle cerebral artery occlusion with or without treatment with a γ-secretase inhibitor (GSI). To investigate the impact of Notch on microglial effector functions, primary mouse microglia and murine BV-2 microglial cell line were exposed to oxygen glucose deprivation or lipopolysaccharide in the presence or absence of GSI. Immunofluorescence labeling, Western blotting, and reverse-transcription polymerase chain reaction were performed to measure microglial activation and production of inflammatory cytokines. The nuclear translocation of nuclear factor-κB in microglia was assessed by immunohistochemistry. The neurotoxic potential of microglia was determined in cocultures. Results— Notch1 antisense mice exhibit significantly lower numbers of activated microglia and reduced proinflammatory cytokine expression in the ipsilateral ischemic cortices compared to non-Tg mice. Microglial activation also was attenuated in Notch1 antisense cultures and in non-Tg cultures treated with GSI. GSI significantly reduced nuclear factor-κB activation and expression of proinflammatory mediators and markedly attenuated the neurotoxic activity of microglia in cocultures. Conclusions— These findings establish a role for Notch signaling in modulating the microglia innate response and suggest that inhibition of Notch might represent a complementary therapeutic approach to prevent reactive gliosis in stroke and neuroinflammation-related degenerative disorders.

The cell fate determinant Numb exists in four alternatively spliced variants that differ in the length of their PTB (phosphotyrosine-binding domain, either lacking or containing an 11 amino acid insertion) and PRR (proline-rich region, either lacking or containing a 48 amino acid insertion). We previously reported that Numb switches from isoforms containing the PTB insertion to isoforms lacking this insertion in neural cultures subjected to stress induced by trophic factor withdrawal. The switch in Numb isoforms enhances the generation of amyloid-β peptide (Aβ), the principle component of senile plaques in Alzheimer's disease (AD). Here we examine the expression of the Numb isoforms in brains from AD patients and triple transgenic (3xTg) AD mice. We found that levels of the Numb isoforms lacking the PTB insertion are significantly elevated in the parietal cortex but not in the cerebellum of AD patients when compared to control subjects. Levels of Numb isoforms lacking the PTB insertion were also elevated in the cortex but not cerebellum of 12 month-old 3xTg AD mice with Aβ deposits compared to younger 3xTg-AD mice and to non-transgenic mice. Exposure of cultured neurons to Aβ resulted in an increase in the levels of Numb isoforms lacking the PTB domain, consistent with a role for Aβ in the aberrant expression of Numb in vulnerable brain regions of AD patients and mice. Collectively, the data show that altered expression of Numb isoforms in vulnerable neurons occurs during AD pathogenesis and suggest a role for Numb in the disease process.

BACKGROUND: Hydrocephalus occurs because of an imbalance of bulk fluid flow in the brain, and aquaporins (AQPs) play pivotal roles in cerebral water movement as essential mediators during edema and fluid accumulation. AQP1 is a water channel found in the choroid plexus (CP), and AQP4 is expressed at the brain-CSF interfaces and astrocytic end feet; excessive fluid accumulation may involve expression of changes in these AQPs during various stages of hydrocephalus. OBJECTIVE: To determine the alterations of CP AQP1 expression in congenital hydrocephalus ; detect hydrocephalus-induced AQP1 expression in the cortical parenchyma, ependyma, and pia mater of hydrocephalic animals; and evaluate AQP4 expression in congenital hydrocephalus through progressive stages of the condition. METHODS: We evaluated differential expression of AQPs 1 and 4 in the congenital hydrocephalus Texas rat at postnatal days 5, 10, and 26 in isolated CP and cortex by enzyme-linked immunosorbent assay, Western blot, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry. RESULTS: The CP exhibited a 34% decrease in AQP1 expression in young hydrocephalic pups (postnatal days 5 and 10), which became normal (postnatal day 26) just before death. With advancing hydrocephalus, expression of AQPs 1 and 4 increased at the brain-CSF interfaces; AQP1 was localized to the endothelium of cortical capillaries with increased AQP4 expression in surrounding astrocytes end feet. AQP1 expression level was increased in the pia mater, with prominent AQP4 expression in the subpial layers. Subependymal capillaries expressed AQP1 in the endothelium, with increasing AQP4 expression in surrounding astrocytes. Hydrocephalic animals (postnatal day 26) had significant nonendothelial (CD34 ― ) AQP1 expression in the septal nucleus of the basal forebrain, an area affected by increased intracranial pressure. CONCLUSION: Biphasic AQP1 expression in the CP with increased AQPs 1 and 4 at the brain-fluid interfaces may indicate compensatory mechanisms to regulate choroidal cerebrospinal fluid secretion and increase parenchymal fluid absorption in the highpressure hydrocephalic condition.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by expanded polyglutamine repeats in the huntingtin (Htt) protein. Mutant Htt may damage and kill striatal neurons by a mechanism involving reduced production of brain-derived neurotrophic factor (BDNF) and increased oxidative and metabolic stress. Because electroconvulsive shock (ECS) can stimulate the production of BDNF and protect neurons against stress, we determined whether ECS treatment would modify the disease process and provide a therapeutic benefit in a mouse model of HD. ECS (50 mA for 0.2 s) or sham treatment was administered once weekly to male N171-82Q Htt mutant mice beginning at 2 months of age. Endpoints measured included motor function, striatal and cortical pathology, and levels of protein chaperones and BDNF. ECS treatment delayed the onset of motor symptoms and body weight loss and extended the survival of HD mice. Striatal neurodegeneration was attenuated and levels of protein chaperones (Hsp70 and Hsp40) and BDNF were elevated in striatal neurons of ECS-treated compared with sham-treated HD mice. Our findings demonstrate that ECS can increase the resistance of neurons to mutant Htt resulting in improved functional outcome and extended survival. The potential of ECS as an intervention in subjects that inherit the mutant Htt gene merits further consideration.

beta1 integrin is a cell surface molecule that is critical for endothelial cell adhesion, migration and survival during angiogenesis. In the present study we employed in vivo and in vitro models to elucidate the role of beta1 integrin in vascular remodelling and stroke outcomes. At 24 h after cerebral ischemia and reperfusion (I/R), the ischemic cortex (ipsilateral area) exhibited modest beta1 integrin immunoreactivity and a robust increase was observed at 72 h. Double-label immunohistochemical analysis for beta1 integrin with neuronal (NeuN), microglial (Iba-1), astrocyte (GFAP), progenitor cell (Ng2) and blood vessel (collagen 4) markers showed that beta1 integrin expression only localized to blood vessels. In vitro studies using cultured endothelial cells and a beta1 integrin blocking antibody confirmed that beta1 integrin is required for endothelial cell migration, proliferation and blood vessel formation. In vivo studies in the cerebral I/R model using the beta1 integrin blocking antibody further confirmed that beta1 integrin signaling is involved in vascular formation and recovery following ischemic stroke. Finally, we found that beta1 integrin is critically involved in functional deficits and survival after a stroke. These results suggest that beta1 integrin plays important roles in neurovascular remodelling and functional outcomes following stroke, and that targeting the beta1 integrin signalling may provide a novel strategy for modulating angiogenesis in ischemic stroke and other pathological conditions.

Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM. Cancer Res; 70(1); 418–27

Stroke is among the three leading causes of death worldwide and the most frequent cause of permanent disability. Brain ischemia induces an inflammatory response involving activated complement fragments. Here we show that i.v. Ig (IVIG) treatment, which scavenges complement fragments, protects brain cells against the deleterious effects of experimental ischemia and reperfusion (I/R) and prevents I/R-induced mortality in mice. Animals administered IVIG either 30 min before ischemia or after 3 h of reperfusion exhibited a 50–60% reduction of brain infarct size and a 2- to 3-fold improvement of the functional outcome. Even a single low dose of IVIG given after stroke was effective. IVIG was protective in the nonreperfusion model of murine stroke as well and did not exert any peripheral effects. Human IgG as well as intrinsic murine C3 levels were significantly higher in the infarcted brain region compared with the noninjured side, and their physical association was demonstrated by immuno-coprecipitation. C5-deficient mice were significantly protected from I/R injury compared with their wild-type littermates. Exposure of cultured neurons to oxygen/glucose deprivation resulted in increased levels of C3 associated with activation of caspase 3, a marker of apoptosis; both signals were attenuated with IVIG treatment. Our data suggest a major role for complement-mediated cell death in ischemic brain injury and the prospect of using IVIG in relatively low doses as an interventional therapy for stroke.

Although a strong correlation between neuroendocrine differentiation and angiogenesis of prostate cancer has been reported, no mechanistic link between the two events has been established. Because neuropeptide calcitonin is secreted by prostate tumors and endothelial cells are known to express calcitonin receptor–like receptor, we examined the potential action of calcitonin on endothelial cells. The presence of calcitonin receptor, calcitonin receptor–like receptor, and receptor activity–modifying proteins in human microvessel endothelial-1 cells was tested by reverse transcriptase-PCR (RT-PCR). The proangiogenic action of calcitonin was examined in several in vitro models of angiogenesis using HMEC-1 cells and also in vivo using dorsal skinfold assays. Calcitonin expression of PC-3M cells was modulated, and its effect on angiogenesis was examined in in vitro as well as in vivo models. The results of RT-PCR and radioligand receptor assays showed the presence of functional calcitonin receptor in HMEC-1 cells. Calcitonin stimulated all phases of angiogenesis through the calcitonin receptor, but its effect on tube morphogenesis by endothelial cells occurred at the concentration of the Kd of calcitonin receptor. Silencing of calcitonin receptor expression in HMEC-1 cells abolished calcitonin-induced tube formation. Vascular endothelial growth factor antibodies attenuated but did not abolish calcitonin-induced tube morphogenesis. PC-3M prostate cancer cells induced angiogenesis in in vivo and in vitro models. Overexpression of calcitonin in PC-3M cells increased their angiogenic activity, whereas the silencing of calcitonin expression abolished it. These results show that prostate tumor–derived calcitonin may play an important role in prostate tumor growth by regulating intratumoral vascularization.

MRI was utilized to probe T2 changes in living brain following exposure of rats to one of ten classical neurotoxicants. Brains were subsequently perfused for classical neuropathology examination. This approach was predicated on the assumption that the T2 changes represent loci of neurotoxicity encompassing those seen using neuropathology techniques. The traditional neurotoxicologic approach of selecting a few arbitrary brain sections is dramatically improved by MRI targeting that can indicate the location(s) at which to collect "smart sections" for subsequent workup. MRI scans can provide the equivalent of 64 coronal sections; the number estimated for full coverage of the rat brain if only traditional neuropathology is utilized. Use of MRI allows each animal to serve as its own control as well as longitudinal observations of the life cycle of the neurotoxic lesion(s) (inception, apex and regression). Optimization of time of sacrifice and selection of an appropriate stain based on MRI-identified brain areas could be greatly enhanced should this approach prove successful. The application of full brain MRI imaging that informs neuropathology offers the potential to dramatically improve detection of neurotoxicity produced by new drugs and facilitate new drug development, review and approval processes, and to qualify an imaging biomarker of neuropathology.

Parkinson's disease (PD) is a progressive motor disease of unknown etiology in the majority of cases. The clinical features of PD emerge due to selective degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNc), which project to the caudate putamen (CPu) where they release DA. In the current in vivo mouse model study, we tested trehalose for its ability to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced damage to DA neurons. Trehalose is a naturally occurring disaccharide present in plants and animals and appears capable of protecting cells against various environmental stresses. The effect of trehalose is likely due to its action as a pharmacological chaperone which promotes protein stability. In the present study, there were four treatment groups: saline only (control); probenecid only; MPTP + probenecid; and trehalose + MPTP + probenecid. MPTP-induced losses in tyrosine hydroxylase and DA transporter immunoreactivity in the ventral midbrain SNc and CPu were significantly reduced by trehalose. Decreases in CPu dopamine levels produced by MPTP were also blocked by trehalose. Microglial activation and astrocytic hypertrophy induced by MPTP were greatly reduced by trehalose, indicating protection against neuroinflammation. These effects are commensurate with the observed trehalose sparing of motor deficits produced by MPTP in this mouse model. Two tight junctional proteins, ZO-1 and occludin, are downregulated following MPTP treatment and trehalose blocks this effect. Likewise, the glucose transporter-1 that is expressed in brain endothelial cells is also protected by trehalose from MPTP-induced down-regulation. This study is the first to demonstrate using fluoro-turoquoise FT gel perfusion techniques, the protection afforded by trehalose from MPTP-induced damage to microvessels and endothelial and suggests that trehalose therapy may have the potential to slow or ameliorate PD pathology.

Several dual-specificity phosphatases (DUSPs) that play key roles in the direct or indirect inactivation of different MAP kinases (MAPKs) have been implicated in human cancers over the past decade. This has led to a growing interest in identifying DUSPs and their specific inhibitors for further testing and validation as therapeutic targets in human cancers. However, the lack of understanding of the complex regulatory mechanisms and cross-talks between MAPK signaling pathways, combined with the fact that DUSPs can act as a double-edged sword in cancer progression, calls for a more careful and thorough investigation. Among the various types of brain cancer, glioblastoma multiforme (GBM) is notorious for its aggressiveness and resistance to current treatment modalities. This has led to the search for new molecular targets, particularly those involving various signaling pathways. DUSPs appear to be a promising target, but much more information on DUSP targets and their effects on GBM is needed before potential therapies can be developed, tested, and validated. This review identifies and summarize the specific roles of DUSP1, DUSP4, DUSP6 and DUSP26 that have been implicated in GBM.

Alzheimer's disease (AD) is an incurable and progressive neurodegenerative senile disorder associated with the brain accumulation of Aβ plaques. Although vaccines that reduce Aβ plaques can control AD, the rationale for their use at the onset of the disease remains debatable. Old humans and mice usually respond poorly to vaccines due to presumably age-related immunological impairments. Here, we report that by modifying vaccines, the poor responsiveness of old mice can be reversed. Unlike the Aβ peptide vaccine, DNA immunizations with the amino-terminal Aβ(1-11) fragment exposed on the surface of HBsAg particles elicit high levels of anti-Aβ antibody both in young and old mice. Importantly, in AD model 3xTgAD mice, the vaccine reduced Aβ plaques, ameliorated cognitive impairments and, surprisingly, significantly increased life span. Hence, we propose that vaccines targeting Aβ(1-11) can efficiently combat AD-induced pathological alterations and provide survival benefit in patients with AD.

Acetazolamide (AZA), used in treatment of early or infantile hydrocephalus, is effective in some cases, while its effect on the choroid plexus (CP) remains ill-defined. The drug reversibly inhibits aquaporin-4 (AQP4), the most ubiquitous "water pore" in the brain, and perhaps modulation of AQP1 (located apically on CP cells) by AZA may reduce cerebrospinal fluid (CSF) production. We sought to elucidate the effect of AZA on AQP1 and fluid flow in CP cell cultures.CP tissue culture from 10-day Sprague-Dawley rats and a TRCSF-B cell line were grown on Transwell permeable supports and treated with 100 μM AZA. Fluid assays to assess direction and extent of fluid flow, and AQP1 expression patterns by immunoblot, Immuncytochemistry (ICC), and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed.Immunoblots and ICC analyses showed a decrease in AQP1 protein shortly after AZA treatment (lowest at 12 h), with transient AQP1 reduction mediated by mRNA expression (lowest at 6 h). Transwell fluid assays indicated a fluid shift at 2 h, before significant changes in AQP1 mRNA or protein levels.Timing of AZA effect on AQP1 suggests the drug alters protein transcription, while affecting fluid flow by a concomitant method. It is plausible that other mechanisms account for these phenomena, as the processes may occur independently.

Acetazolamide (AZA), used in treatment of early or infantile hydrocephalus, is effective in some cases, while its effect on the choroid plexus (CP) remains ill-defined. The drug reversibly inhibits aquaporin-4 (AQP4), the most ubiquitous “water pore” in the brain, and perhaps modulation of AQP1 (located apically on CP cells) by AZA may reduce cerebrospinal fluid (CSF) production. We sought to elucidate the effect of AZA on AQP1 and fluid flow in CP cell cultures.

Because the olfactory system plays a major role in food consumption, and because “food addiction” and associated morbidities have reached epidemic proportions, we tested the hypothesis that dietary energy restriction can modify adverse effects of cocaine on behavior and olfactory cellular and molecular plasticity. Mice maintained on an alternate day fasting (ADF) diet exhibited increased baseline locomotion and increased cocaine-sensitized locomotion during cocaine conditioning, despite no change in cocaine conditioned place preference, compared to mice fed ad libitum. Levels of dopamine and its metabolites in the olfactory bulb (OB) were suppressed in mice on the ADF diet compared to mice on the control diet, independent of acute or chronic cocaine treatment. The expression of several enzymes involved in dopamine metabolism including tyrosine hydroxylase, monoamine oxidases A and B (MAOA), and catechol-O-methyltransferase were significantly reduced in OBs of mice on the ADF diet. Both acute and chronic administration of cocaine suppressed the production of new OB cells, and this effect of cocaine was attenuated in mice on the ADF diet. Cocaine administration to mice on the control diet resulted in up-regulation of OB genes involved in mitochondrial energy metabolism, synaptic plasticity, cellular stress responses, and calcium- and cyclic AMP-mediated signaling, whereas multiple olfactory receptor genes were down-regulated by cocaine treatment. ADF abolished many of the effects of cocaine on OB gene expression. Our findings reveal that dietary energy intake modifies the neural substrates underlying some of the behavioral and physiological responses to repeated cocaine treatment, and also suggest novel roles for the olfactory system in addiction. The data further suggest that modification of dietary energy intake could provide a novel potential approach to addiction treatments.

Because the olfactory system plays a major role in food consumption, and because 'food addiction' and associated morbidities have reached epidemic proportions, we tested the hypothesis that dietary energy restriction can modify adverse effects of cocaine on behavior and olfactory cellular and molecular plasticity. Mice maintained on an alternate day fasting (ADF) diet exhibited increased baseline locomotion and increased cocaine-sensitized locomotion during cocaine conditioning, despite no change in cocaine conditioned place preference, compared with mice fed ad libitum. Levels of dopamine and its metabolites in the olfactory bulb (OB) were suppressed in mice on the ADF diet compared with mice on the control diet, independent of acute or chronic cocaine treatment. The expression of several enzymes involved in dopamine metabolism including tyrosine hydroxylase, monoamine oxidases A and B, and catechol-O-methyltransferase were significantly reduced in OBs of mice on the ADF diet. Both acute and chronic administration of cocaine suppressed the production of new OB cells, and this effect of cocaine was attenuated in mice on the ADF diet. Cocaine administration to mice on the control diet resulted in up-regulation of OB genes involved in mitochondrial energy metabolism, synaptic plasticity, cellular stress responses, and calcium- and cAMP-mediated signaling, whereas multiple olfactory receptor genes were down-regulated by cocaine treatment. ADF abolished many of the effects of cocaine on OB gene expression. Our findings reveal that dietary energy intake modifies the neural substrates underlying some of the behavioral and physiological responses to repeated cocaine treatment, and also suggest novel roles for the olfactory system in addiction. The data further suggest that modification of dietary energy intake could provide a novel potential approach to addiction treatments.

Wound healing is a complex process involving intrinsic dermal and epidermal cells, and infiltrating macrophages and leukocytes. Excessive oxidative stress and associated inflammatory processes can impair wound healing, and antioxidants have been reported to improve wound healing in animal models and human subjects. Uric acid (UA) is an efficient free radical scavenger, but has a very low solubility and poor tissue penetrability. We recently developed novel UA analogs with increased solubility and excellent free radical-scavenging properties and demonstrated their ability to protect neural cells against oxidative damage. Here we show that the uric acid analog (6, 8 dithio-UA, but not equimolar concentrations of UA or 1, 7 dimethyl-UA) modified the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in ways consistent with enhancement of the wound healing functions of all three cell types. We further show that 6, 8 dithio-UA significantly accelerates the wound healing process when applied topically (once daily) to full-thickness wounds in mice. Levels of Cu/Zn superoxide dismutase were increased in wound tissue from mice treated with 6, 8 dithio-UA compared to vehicle-treated mice, suggesting that the UA analog enhances endogenous cellular antioxidant defenses. These results support an adverse role for oxidative stress in wound healing and tissue repair, and provide a rationale for the development of UA analogs in the treatment of wounds and for modulation of angiogenesis in other pathological conditions.

Mitochondrial dysfunction and reactive oxygen species (ROS) production are at the heart of the aging process and are thought to underpin age-related diseases. Mitochondria are not only the primary energy-generating system but also the dominant cellular source of metabolically derived ROS. Recent studies unravel the existence of mechanisms that serve to modulate the balance between energy metabolism and ROS production. Among these is the regulation of proton conductance across the inner mitochondrial membrane that affects the efficiency of respiration and heat production. The field of mitochondrial respiration research has provided important insight into the role of altered energy balance in obesity and diabetes. The notion that respiration and oxidative capacity are mechanistically linked is making significant headway into the field of aging and age-related diseases. Here we review the regulation of cellular energy and ROS balance in biological systems and survey some of the recent relevant studies that suggest that respiratory adaptation and thermodynamics are important in aging and age-related diseases.

Claudin proteins are frequently overexpressed in various tumors such as breast, prostate and ovarian cancer. While their functions in cancer have not been completely elucidated, roles in survival, adhesion, and invasion have been suggested. In order to clarify the roles of claudins in ovarian cancer, we have performed gene expression profiling of ovarian surface epithelial cells overexpressing claudin-4 and compared the expression patterns to the parental, non-expressing cells. Claudin-4 expression leads to the differential expression of several genes, including many that have previously been implicated in angiogenesis. In particular, angiogenic cytokines, such as IL-8, were found elevated while genes of the angiostatic interferon pathway were found down-regulated. In vitro assays show that claudin-4-expressing cells produce factors that can stimulate angiogenesis as measured by tube formation and migration in HUVEC cells. In addition, an in vivo mouse dorsal skinfold assay confirms that cells expressing claudin-4 secrete factors that can mediate angiogenesis in the dorsal skin of mice. Our data suggest a novel function for claudin-4 in cancer and provide an additional rationale for its common overexpression in human tumors.

IInteractions between laminins and integrin receptors hold neural stem cells in place at the ventricular surface of embryonic brain. Transient disruption leads to abnormal stem cell divisions and permanent cortical malformation., During embryogenesis, the neural stem cells (NSC) of the developing cerebral cortex are located in the ventricular zone (VZ) lining the cerebral ventricles. They exhibit apical and basal processes that contact the ventricular surface and the pial basement membrane, respectively. This unique architecture is important for VZ physical integrity and fate determination of NSC daughter cells. In addition, the shorter apical process is critical for interkinetic nuclear migration (INM), which enables VZ cell mitoses at the ventricular surface. Despite their importance, the mechanisms required for NSC adhesion to the ventricle are poorly understood. We have shown previously that one class of candidate adhesion molecules, laminins, are present in the ventricular region and that their integrin receptors are expressed by NSC. However, prior studies only demonstrate a role for their interaction in the attachment of the basal process to the overlying pial basement membrane. Here we use antibody-blocking and genetic experiments to reveal an additional and novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal INM and alterations in the orientation of NSC divisions. We found that these defects were also observed in laminin α2 deficient mice. More detailed analyses using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex. Collectively, these data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSCs to the ventricular surface and maintaining the physical integrity of the neocortical niche, with even transient perturbations resulting in long-lasting cortical defects., Author Summary The developing cerebral cortex contains bipolar neural stem cells that span the cortical layers between the inner ventricular surface and the outer pial surface of the embryonic brain. The nuclei of these cells remain near the ventricular cavity, a microenvironment or niche thought to provide vital signals. It is not known how this inner end of the bipolar stem cell is held in place, or what would happen if its attachment to the inner surface were lost. Genetic manipulation can be used to disrupt candidate molecules involved in this adhesion, but this will affect all adhesion points and complicate the results. We have therefore developed an approach to target the stem cell attachments specifically at the ventricular surface by placing blocking antibodies directly into the ventricles of mouse embryos and then expressing fluorescent markers in the stem cells to see the effects of losing this attachment in living tissue. We examined laminins and integrins, whose expression and properties make them excellent candidates. Blocking integrin signalling by antibody application caused the inner end of the stem cells to rapidly detach and then undergo aberrant cell division. We also showed that a transient block of integrins (for

The endoplasmic reticulum (ER) is a key organelle regulating intracellular Ca(2+) homeostasis. Oxidants and mitochondria-derived free radicals can target ER-based Ca(2+) regulatory proteins and cause uncontrolled Ca(2+) release that may contribute to protracted ER stress and apoptosis. Several ER stress proteins have been suggested to counteract the deregulation of ER Ca(2+) homeostasis and ER stress. Here we showed that knockdown of Herp, an ubiquitin-like domain containing ER stress protein, renders PC12 and MN9D cells vulnerable to 1-methyl-4-phenylpyridinium-induced cytotoxic cell death by a mechanism involving up-regulation of CHOP expression and ER Ca(2+) depletion. Conversely, Herp overexpression confers protection by blocking 1-methyl-4-phenylpyridinium-induced CHOP up-regulation, ER Ca(2+) store depletion, and mitochondrial Ca(2+) accumulation in a manner dependent on a functional ubiquitin-proteasomal protein degradation pathway. Deletion of the ubiquitin-like domain of Herp or treatment with a proteasomal inhibitor abolished the central function of Herp in ER Ca(2+) homeostasis. Thus, elucidating the underlying molecular mechanism(s) whereby Herp counteracts Ca(2+) disturbances will provide insights into the molecular cascade of cell death in dopaminergic neurons and may uncover novel therapeutic strategies to prevent and ameliorate Parkinson disease progression.

Increased bioenergetics demand can stimulate compensatory increases in glucose metabolism. We previously reported that neural cells expressing the brain uncoupling protein UCP4 exhibit enhanced dependency on glucose for support of cellular bioenergetics and survival. The switch from oxidative toward glycolytic metabolism reduces the production of toxic reactive oxygen species (ROS) and increases cellular resistance to toxicity induced by 3-nitropropionic acid, a mitochondrial complex II inhibitor that compromises cellular bioenergetics. In this study we elucidate the underlying mechanism whereby expression of UCP4 promotes bioenergetics adaptation and cell survival. We found that activation of extracellular signal-regulated kinases (ERKs) is necessary and sufficient for the increased dependency on glucose utilization. Pharmacological inhibition of ERKs not only abrogated bioenergetics adaptation but also reduced the activation of cAMP-responsive element-binding (CREB) protein suggesting that CREB protein signaling contributes in part to UCP4-dependent cell death rescue from 3-nitropropionic acid-induced toxicity. We also demonstrated that activation of ERKs by growth factors ameliorated the bioenergetics compromise and reduced cellular toxicity induced by 3-nitropropionic acid. Collectively, our results support the involvement of ERKs in UCP4 dependent bioenergetics adaptation and cell survival.

Regular exercise can counteract the adverse effects of aging on the musculoskeletal and cardiovascular systems. In males the normal aging process is associated with reductions in testosterone production’(Matsumoto 2002); (synder 2001) and impaired spermatogenesis, but the underlying mechanisms and their potential modification by exercise are unknown. Here we report that lifelong regular exercise (running) protects the testes against maturation as well as age related changes, and that this effect of running is associated with decreased amounts of oxidative damage to proteins, lipids and DNA in spermatogenic and Leydig cells. Six month-old male mice were divided into a sedentary group and a group which ran an average of 1.75 km/day, until the mice reached the age of 20 months. Seminiferous tubules of runners exhibited a full complement of cells at different stages of the spermatogenic process and a clear central lumen with large numbers of spermatozoa, in contrast to sedentary mice which exhibited disorganized spermatogenic cells and lacked spermatocytes in a central lumen. Levels of protein carbonyls, nitrotyrosine, lipid peroxidation products and oxidatively modified DNA were significantly greater in spermatogenic and Leydig cells of sedentary mice compared to runners. These findings suggest that lifelong regular exercise suppresses aging of the testes by a mechanism that involves reduced oxidative damage to spermatogenic and Leydig cells.

Although neurologic sequelae of acute kidney injury (AKI) are well described, the pathogenesis of acute uremic encephalopathy is poorly understood. This study examined the short-term effect of ischemic AKI on inflammatory and functional changes of the brain in mice by inducing bilateral renal ischemia for 60 min and studying the brains 24 h later. Compared with sham mice, mice with AKI had increased neuronal pyknosis and microgliosis in the brain. AKI also led to increased levels of the proinflammatory chemokines keratinocyte-derived chemoattractant and G-CSF in the cerebral cortex and hippocampus and increased expression of glial fibrillary acidic protein in astrocytes in the cortex and corpus callosum. In addition, extravasation of Evans blue dye into the brain suggested that the blood-brain barrier was disrupted in mice with AKI. Because liver failure also leads to encephalopathy, ischemic liver injury was induced in mice with normal renal function; neuronal pyknosis and glial fibrillary acidic protein expression were not increased, suggesting differential effects on the brain depending on the organ injured. For evaluation of the effects of AKI on brain function, locomotor activity was studied using an open field test. Mice subjected to renal ischemia or bilateral nephrectomy had moderate to severe declines in locomotor activity compared with sham-operated mice. These data demonstrate that severe ischemic AKI induces inflammation and functional changes in the brain. Targeting these pathways could reduce morbidity and mortality in critically ill patients with severe AKI.

The Notch signaling pathway is critically involved in cell fate decisions during development of many tissues and organs. In the present study we employed in vivo and cell culture models to elucidate the role of Notch signaling in wound healing. The healing of full-thickness dermal wounds was significantly delayed in Notch antisense transgenic mice and in normal mice treated with gamma-secretase inhibitors that block proteolytic cleavage and activation of Notch. In contrast, mice treated with a Notch ligand Jagged peptide showed significantly enhanced wound healing compared to controls. Activation or inhibition of Notch signaling altered the behaviors of cultured vascular endothelial cells, keratinocytes and fibroblasts in a scratch wound healing model in ways consistent with roles for Notch signaling in wound healing functions all three cell types. These results suggest that Notch signaling plays important roles in wound healing and tissue repair, and that targeting the Notch pathway might provide a novel strategy for treatment of wounds and for modulation of angiogenesis in other pathological conditions.

The innate immune system senses the invasion of pathogenic microorganisms and tissue injury through Toll-like receptors (TLR), a mechanism thought to be limited to immune cells. We now report that neurons express several TLRs, and that the levels of TLR2 and -4 are increased in neurons in response to IFN-γ stimulation and energy deprivation. Neurons from both TLR2 knockout and -4 mutant mice were protected against energy deprivation-induced cell death, which was associated with decreased activation of a proapoptotic signaling cascade involving jun N-terminal kinase and the transcription factor AP-1. TLR2 and -4 expression was increased in cerebral cortical neurons in response to ischemia/reperfusion injury, and the amount of brain damage and neurological deficits caused by a stroke were significantly less in mice deficient in TLR2 or -4 compared with WT control mice. Our findings establish a proapoptotic signaling pathway for TLR2 and -4 in neurons that may render them vulnerable to ischemic death.

The expression of human (h) calcitonin (CT) and its receptor (CTR) is localized to basal epithelium in benign prostates but is distributed in whole epithelium of malignant prostates. Moreover, the abundance of hCT and CTR mRNA in primary prostate tumors positively correlates with the tumor grade. We tested the hypothesis that the modulation of endogenous hCT expression of prostate cancer (PC) cell lines alters their oncogenicity. The effect of modulation of hCT expression on oncogenic characteristics was examined in LNCaP and PC-3M cell lines. The endogenous hCT expression was modulated using either constitutively active expression vector containing hCT cDNA or anti-hCT hammerhead ribozymes. The changes in the oncogenicity of cell sublines was assessed with cell proliferation assays, invasion assays, colony formation assays, and in vivo growth in athymic nude mice. Up-regulation of hCT in PC-3M cells and or enforced hCT expression in LNCaP cells dramatically enhanced their oncogenic characteristics. In contrast, the down-regulation of hCT in PC-3M cells led to a dramatic decline in their oncogenicity. These results, when combined with our other results, that the expression of hCT in primary PCs increase with tumor grade, suggest an important role for hCT in the progression of PC to a metastatic phenotype.

Calcitonin (CT) is synthesized and secreted in prostate epithelium, and its secretion from malignant prostates is several folds higher than that in benign prostates. CT receptor (CTR) is expressed in malignant prostate epithelium, and its activation increases invasiveness of prostate cancer (PC) cells via activation of protein kinase A. Since the role of urokinase-type plasminogen activator (uPA) in invasion of PC has been established, we tested the hypothesis that CT increases invasion of PC cells by stimulating uPA secretion from PC cells. Exogenously added CT stimulated the secretion of uPA from PC-3M cells in a dose-dependent manner, which was blocked by Rp.cAMP, a competitive inhibitor of protein kinase A. CT stimulated the secretion of MMP-2 and MMP-9 from PC-3M cells, and also increased their invasiveness. Both these actions of CT were blocked by uPA-neutralizing antibodies. Immunofluorescence studies with PC-3M cells suggest that CT stimulated redistribution of cellular uPA to focal adhesion sites, which was further confirmed by co-immunoprecipitation of uPA with focal adhesion kinase (FAK) in response to CT. These results suggest that CT increases invasiveness of PC cells by stimulating PKA-mediated uPA secretion and by redirecting the secreted uPA to focal adhesion sites. The results also suggest that uPA may, at least in part, mediate proinvasive actions of CT on PC cells by stimulating the secretion of gelatinases and degradation of focal adhesion sites.