Heat shock protein 90␣ (Hsp90␣) is a ubiquitously expressed molecular chaperone that is essential for eukaryotic homeostasis. Hsp90␣ can also be secreted extracellularly, where it has been shown to be involved in tumor metastasis. Extracellular Hsp90␣ interacts with and promotes the proteolytic activity of matrix metalloproteinase-2 (MMP-2). However, the regulatory mechanism of Hsp90␣ on MMP-2 activity is still unknown. Here we show that Hsp90␣ stabilizes MMP-2 and protects it from degradation in tumor cells. Further investigation reveals that this stabilization effect is isoform-specific, ATP-independent, and mediated by the interaction between the Hsp90␣ middle domain and the MMP-2 C-terminal hemopexin domain. Moreover, this mechanism also applies to endothelial cells that secrete more Hsp90␣ in their proliferating status. Furthermore, endothelial cell transmigration, Matrigel plug, and tumor angiogenesis assays demonstrate that extracellular Hsp90␣ promotes angiogenesis in an MMP-2-dependent manner. In sum, this study provides new insights into the molecular mechanism of how Hsp90␣ regulates its extracellular client proteins and also reveals for the first time the function of extracellular Hsp90␣ in promoting tumor angiogenesis.Heat shock protein 90 (Hsp90) 2 is an ATP-dependent molecular chaperone that is ubiquitously expressed and essential for cell viability (1). Unlike other types of chaperones, Hsp90 is not required for the biogenesis of most polypeptides but instead functions in the maintenance of the active state of several conformationally labile signaling proteins (2-4). Many of the Hsp90 client proteins are mutated, chimeric, or overexpressed oncogenic proteins (3). Therefore, the chaperoning function of Hsp90 is subverted to a biochemical buffer for genetic lesions in tumor cells, facilitating the malignant transformations of the cells (3). Hsp90 has emerged as a promising target for cancer therapy (5).There are two isoforms of Hsp90 in the cytosol, referred to as Hsp90␣ and Hsp90␤ (6). Intriguingly, the Hsp90␣ isoform also exists extracellularly (7). Recent studies indicate that extracellular Hsp90␣ is significantly correlated with tumor invasiveness and metastasis (8), and the antibody or impermeable inhibitor of Hsp90␣ can suppress tumor metastasis efficiently in mouse models (9 -11). Furthermore, Hsp90␣ can be detected in the blood of cancer patients, and the level of Hsp90␣ is positively associated with tumor malignancy (9). In addition to tumor cells, extracellular Hsp90␣ has also been identified in neuron cells, dermal fibroblasts, keratinocyte, macrophages, and epithelial cells and participates in neuronal cell migration, wound healing, and viral and bacteria infections (7).Accumulating evidence indicates that extracellular Hsp90␣ plays important roles in both physiological and pathological processes, especially in tumor progression (7). However, the molecular mechanism of how extracellular Hsp90␣ functions is still largely unknown (12). Eustace et al. (8) reported that extracellular Hsp90...

Water channel aquaporin-1 (AQP1) is expressed at epithelial cell plasma membranes in renal proximal tubules and thin descending limb of Henle. Recently, AQP1 was reported to interact with b-catenin. Here we investigated the relationship between AQP1 and Wnt signaling in in vitro and in vivo models of autosomal dominant polycystic kidney disease (PKD). AQP1 overexpression decreased b-catenin and cyclinD1 expression, suggesting down-regulation of Wnt signaling, and coimmunoprecipitation showed AQP1 interaction with bcatenin, glycogen synthase kinase 3b, LRP6, and Axin1. AQP1 inhibited cyst development and promoted branching in matrix-grown MDCK cells. In embryonic kidney cultures, AQP1 deletion increased cyst development by up to ∼40%. Kidney size and cyst number were significantly greater in AQP1-null PKD mice than in AQP1-expressing PKD mice, with the difference mainly attributed to a greater number of proximal tubule cysts. Biochemical analysis revealed decreased b-catenin phosphorylation and increased b-catenin expression in AQP1-null PKD mice, suggesting enhanced Wnt signaling. These results implicate AQP1 as a novel determinant in renal cyst development that may involve inhibition of Wnt signaling by an AQP1-macromolecular signaling

Safflower has long been used to treat cerebrovascular diseases in China. We previously reported that kaempferol derivatives of safflower can bind DJ-1, a protein associated with Parkinson's disease (PD), and flavonoid extract of safflower exhibited neuroprotective effects in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of PD. In this study, a standardized safflower flavonoid extract (SAFE) was isolated from safflower and mainly contained flavonoids. Two marker compounds of SAFE, kaempferol 3-O-rutinoside and anhydrosafflor yellow B, were proven to suppress microtubule destabilization and decreased cell area, respectively. We confirmed that SAFE in dripping pill form could improve behavioural performances in a 6-hydroxydopamine (6-OHDA)-induced rat model of PD, partially via the suppression of α-synuclein overexpression or aggregation, as well as the suppression of reactive astrogliosis. Using an MRI tracer-based method, we found that 6-OHDA could change extracellular space (ECS) diffusion parameters, including a decrease in tortuosity and the rate constant of clearance and an increase in the elimination half-life of the tracer in the 6-OHDA-lesioned substantia nigra. SAFE treatment could partially inhibit the changes in ECS diffusion parameters, which might provide some information about neuronal loss and astrocyte activation. Consequently, our results indicate that SAFE is a potential therapeutic herbal product for treatment of PD.Parkinson's disease (PD) is the second most common disorder of the central nervous system (CNS), and its incidence is increasing among people over the age of 60 years 1 . PD is pathologically characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the formation of cytoplasmic inclusion bodies; however, the aetiology of PD remains elusive. The clinical features of PD include muscular rigidity, resting tremor, bradykinesia, and postural instability. By the time patients are diagnosed with PD, approximately 80% of the striatal dopamine terminals have been lost 2 , and destruction of terminal fields may precede cell body loss in the SN 3 . In rats, the unilateral intracerebral injection of 6-hydroxydopamine (6-OHDA) results in a selective degeneration of dopaminergic neurons, and this is a widely used animal model of PD. 6-OHDA induces a neurodegenerative process in the nigrostriatal system through the inhibition of mitochondrial complex function, which can lead to the induction of oxidative stress, inflammation 4-6 , abnormal protein aggregation 7,8 , elevated iron levels 9 and ultimately cell death. Dopamine replacement therapy remains the first line strategy in PD treatment. However, its effectiveness cannot modify the progression of the neurodegenerative process. Additionally, dopamine replacement therapy is associated with side-effects that include fluctuations in motor response and dyskinesia 10 . Increasing attention 1

Urea transporters (UTs) are a family of membrane channel proteins that are specifically permeable to urea and play an important role in intrarenal urea recycling and in urine concentration. Using an erythrocyte osmotic lysis assay, we screened a small-molecule library for inhibitors of UT-facilitated urea transport. A novel class of thienoquinolin UT-B inhibitors were identified, of which PU-14 had potent inhibition activity on human, rabbit, rat, and mouse UT-B. The half-maximal inhibitory concentration of PU-14 on rat UT-B-mediated urea transport was ∼0.8 μmol/l, and it did not affect urea transport in mouse erythrocytes lacking UT-B but inhibited UT-A-type urea transporters, with 36% inhibition at 4 μmol/l. PU-14 showed no significant cellular toxicity at concentrations up to its solubility limit of 80 μmol/l. Subcutaneous delivery of PU-14 (at 12.5, 50, and 100 mg/kg) to rats caused an increase of urine output and a decrease of the urine urea concentration and subsequent osmolality without electrolyte disturbances and liver or renal damages. This suggests that PU-14 has a diuretic effect by urea-selective diuresis. Thus, PU-14 or its analogs might be developed as a new diuretic to increase renal fluid clearance in diseases associated with water retention without causing electrolyte imbalance. PU-14 may establish 'chemical knockout' animal models to study the physiological functions of UTs.