CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

CRIPTO (CR-1, TDGF1) is a cell surface/secreted oncoprotein actively involved in development and cancer. Here, we report that high expression of CRIPTO correlates with poor survival in stratified risk groups of prostate cancer (PCa) patients. CRIPTO and its signaling partner glucose-regulated protein 78 (GRP78) are highly expressed in PCa metastases and display higher levels in the metastatic ALDHhigh sub-population of PC-3M-Pro4Luc2 PCa cells compared with non-metastatic ALDHlow. Coculture of the osteotropic PC-3M-Pro4Luc2 PCa cells with differentiated primary human osteoblasts induced CRIPTO and GRP78 expression in cancer cells and increases the size of the ALDHhigh sub-population. Additionally, CRIPTO or GRP78 knockdown decreases proliferation, migration, clonogenicity and the size of the metastasis-initiating ALDHhigh sub-population. CRIPTO knockdown reduces the invasion of PC-3M-Pro4Luc2 cells in zebrafish and inhibits bone metastasis in a preclinical mouse model. These results highlight a functional role for CRIPTO and GRP78 in PCa metastasis and suggest that targeting CRIPTO/GRP78 signaling may have significant therapeutic potential.

Tumor microenvironment heterogeneity: challenges and opportunities.

The tumor microenvironment (TME) has been recognized as an integral component of malignancies in breast and prostate tissues, contributing in confounding ways to tumor progression, metastasis, therapy resistance and disease recurrence. Major components of the TME are immune cells, fibroblasts, pericytes, endothelial cells, mesenchymal stroma/stem cells (MSCs), and extracellular matrix (ECM) components. Herein, we discuss the molecular and cellular heterogeneity within the TME and how this presents unique challenges and opportunities for treating breast and prostate cancers.

Activin A/BMP2 chimera AB235 drives efficient redifferentiation of long term cultured autologous chondrocytes.

Autologous chondrocyte implantation (ACI) depends on the quality and quantity of implanted cells and is hindered by the fact that chondrocytes cultured for long periods of time undergo dedifferentiation. Here we have developed a reproducible and efficient chondrogenic protocol to redifferentiate chondrocytes isolated from osteoarthritis (OA) patients. We used morphological, histological and immunological analysis together with a RT-PCR detection of collagen I and collagen II gene expression to show that chondrocytes isolated from articular cartilage biopsies of patients and subjected to long-term culture undergo dedifferentiation and that these cells can be redifferentiated following treatment with the chimeric Activin A/BMP2 ligand AB235. Examination of AB235-treated cell pellets in both in vitro and in vivo experiments revealed that redifferentiated chondrocytes synthesized a cartilage-specific extracellular matrix (ECM), primarily consisting of vertically-orientated collagen fibres and cartilage-specific proteoglycans. AB235-treated cell pellets also integrated into the surrounding subcutaneous tissue following transplantation in mice as demonstrated by their dramatic increase in size while non-treated control pellets disintegrated upon transplantation. Thus, our findings describe an effective protocol for the promotion of redifferentiation of autologous chondrocytes obtained from OA patients and the formation of a cartilage-like ECM that can integrate into the surrounding tissue in vivo.

Blockade of Cripto binding to cell surface GRP78 inhibits oncogenic Cripto signaling via MAPK/PI3K and Smad2/3 pathways.

Cripto is a developmental oncoprotein that signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/Akt and Smad2/3 pathways. However, the molecular basis for Cripto coupling to these pathways during embryogenesis and tumorigenesis is not fully understood. In this regard, we recently demonstrated that Cripto forms a cell surface complex with the HSP70 family member glucose-regulated protein-78 (GRP78). Here, we provide novel functional evidence demonstrating that cell surface GRP78 is a necessary mediator of Cripto signaling in human tumor, mammary epithelial and embryonic stem cells. We show that targeted disruption of the cell surface Cripto/GRP78 complex using shRNAs or GRP78 immunoneutralization precludes Cripto activation of MAPK/PI3K pathways and modulation of activin-A, activin-B, Nodal and transforming growth factor-beta1 signaling. We further demonstrate that blockade of Cripto binding to cell surface GRP78 prevents Cripto from increasing cellular proliferation, downregulating E-Cadherin, decreasing cell adhesion and promoting pro-proliferative responses to activin-A and Nodal. Thus, disrupting the Cripto/GRP78 binding interface blocks oncogenic Cripto signaling and may have important therapeutic value in the treatment of cancer.

Dynamics of the nucleated polymerization model of prion replication.

The disease process for transmissible spongiform encephalopathies (TSEs), in one way or another, involves the conversion of a predominantly alpha-helical normal host-coded prion protein (PrP(C)) to an abnormally folded (predominantly beta sheet) protease resistant isoform (PrP(Sc)). Several alternative mechanisms have been proposed for this auto-catalytic process. Here the dynamical behavior of one of these models, the nucleated polymerization model, is studied by Monte Carlo discrete-event simulation of the explicit conversion reactions. These simulations demonstrate the characteristic dynamical behavior of this model for prion replication. Using estimates for the reaction rates and concentrations, time courses are estimated for concentration of PrP(Sc), PrP(Sc) aggregates, and PrP(C) as well as size distributions for the aggregates. The implications of these dynamics on protein misfolding cyclic amplification (PMCA) is discussed.

Hidden markov model for competitive binding and chain elongation.

Many chemical systems of interest consist of sets of reactions that contain iterated sequences that elongate a molecular chain. For example, such sets of reactions are commonly found in the transcription of DNA or the translation of RNA. However, there are competitive reactions that can prematurely terminate the chain-elongation process. A hidden Markov method appropriate for modeling chains of reactions with competitive processes (i.e., premature chain termination) is developed. The method is an extension of a hidden Markov model suggested by Gibson and Bruck (J. Phys. Chem. A 2000, 104, 1876). The equivalence between results using this method and results from simulation of a system employing the full set of reactions will be demonstrated (Gillespie, D. T. Markov Processes: An Introduction for Physical Scientists; Academic: San Diego, CA, 1992). Examples of its use are shown for several test problems. As an example of a practical application, a comparison among results for a model of terminal modification of ligand-aggregated receptors (Hlavacek, W.; Redondo, A.; Wofsy, C.; Goldstein, B. Bull. Math. Biol. 2002, 64, 887) simulated by ordinary differential equations, the full set of reactions, and the hidden Markov method are shown.

Antagonism of activin by inhibin and inhibin receptors: a functional role for betaglycan-glycan.

Activin and inhibin research has provided important insight into reproductive physiology as well as many areas involving regulation of cell growth, differentiation and function. Progress in understanding the roles of these hormones in various cell and tissue types has been complimented by novel discoveries at the molecular level that have shed light on ligand/receptor interactions, signaling mechanisms and regulation. While the receptors and signaling pathway for activin are now well characterized, the molecular basis for inhibin action has remained relatively unclear. Here we summarize recent advances in understanding inhibin's mode of action focusing on our recent identification of betaglycan-glycan as an inhibin co-receptor capable of mediating inhibin action.

Betaglycan binds inhibin and can mediate functional antagonism of activin signalling.

Activins and inhibins, structurally related members of the TGF-beta superfamily of growth and differentiation factors, are mutually antagonistic regulators of reproductive and other functions. Activins bind specific type II receptor serine kinases (ActRII or IIB) to promote the recruitment and phosphorylation of the type I receptor serine kinase, ALK4 (refs 7-9), which then regulates gene expression by activating Smad proteins. Inhibins also bind type II activin receptors but do not recruit ALK4, providing a competitive model for the antagonism of activin by inhibin. Inhibins fail to antagonize activin in some tissues and cells, however, suggesting that additional components are required for inhibin action. Here we show that the type III TGF-beta receptor, betaglycan, can function as an inhibin co-receptor with ActRII. Betaglycan binds inhibin with high affinity and enhances binding in cells co-expressing ActRII and betaglycan. Inhibin also forms crosslinked complexes with both recombinant and endogenously expressed betaglycan and ActRII. Finally, betaglycan confers inhibin sensitivity to cell lines that otherwise respond poorly to this hormone. The ability of betaglycan to facilitate inhibin antagonism of activin provides a variation on the emerging roles of proteoglycans as co-receptors modulating ligand-receptor sensitivity, selectivity and function.

Identification of a binding site on the type II activin receptor for activin and inhibin.

Type II activin receptors (ActRII and ActRIIB) are single-transmembrane domain serine/threonine kinase receptors that bind activin to initiate the signaling and cellular responses triggered by this hormone. Inhibin also binds type II activin receptors and antagonizes many activin effects. Here we describe alanine scanning mutagenesis of the ActRII extracellular domain. We identify a cluster of three hydrophobic residues (Phe(42), Trp(60), and Phe(83)) that, when individually mutated to alanine in the context of the full-length receptor, cause the disruption of activin and inhibin binding to ActRII. Each of the alanine-substituted ActRII mutants retaining activin binding maintains the ability to form cross-linked complexes with activin and supports activin cross-linking to the type I activin receptor ALK4. Unlike wild-type ActRII, the three mutants unable to bind activin do not cause an increase in activin signaling when transiently expressed in a corticotroph cell line. Together, our results implicate these residues in forming a critical binding surface on ActRII required for functional interactions with both activin and inhibin. This first identification of a transforming growth factor-beta family member binding site may provide a general basis for characterizing binding sites for other members of the superfamily.

AKAP15 anchors cAMP-dependent protein kinase to brain sodium channels.

The voltage-sensitive sodium channel is regulated by cAMP-dependent protein kinase (PKA) phosphorylation. Using purified preparations of rat brain sodium channels, we have shown that the alpha subunit was phosphorylated by a co-purifying protein kinase. The co-purifying kinase was stimulated by cAMP and phosphorylated PKA substrate peptides. Both the regulatory and catalytic subunits of PKA were detected by immunoblotting in purified sodium channel preparations. Bound PKA was immunoprecipitated with anti-SP19 antibodies directed against the sodium channel alpha subunit. PKA bound to sodium channels phosphorylated the sodium channel alpha subunit on the same four serine residues as observed with exogenously added PKA, indicating that association with the sodium channel does not restrict the sites of phosphorylation. Analysis of proteins with high affinity for the type II alpha regulatory subunit of PKA in a gel overlay assay identified a 15-kDa cAMP-dependent protein kinase-anchoring protein (AKAP) in these preparations. Determination of its amino acid sequence by mass spectrometry revealed two peptides identical to AKAP15, a recently described AKAP that targets PKA to skeletal muscle calcium channels. The co-purifying AKAP was also immunoreactive with antibodies generated against AKAP15, and antibodies directed against AKAP15 co-immunoprecipitated the sodium channel. Our results indicate that PKA is bound to brain sodium channels through interaction with AKAP15. Association of AKAP15 with both skeletal muscle calcium channels and brain sodium channels suggests that it may have broad specificity in targeting PKA to ion channels for regulation.

Regulation of ion channels by cAMP-dependent protein kinase and A-kinase anchoring proteins.

Subcellular targeting of the cAMP-dependent protein kinase is achieved, in part, through association with A-kinase anchoring proteins (AKAPs). Recent evidence suggests that specific AKAPs direct the kinase to submembrane sites to facilitate phosphorylation and modulation of a variety of ion channels. A new membrane-anchored AKAP targets cAMP-dependent protein kinase to calcium channels and enhances their regulation in multiple cell types.

Primary structure and function of an A kinase anchoring protein associated with calcium channels.

Rapid, voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A kinase anchoring protein (AKAP). Here we report the isolation, primary sequence determination, and functional characterization of AKAP15, a lipid-anchored protein of 81 amino acid residues with a single amphipathic helix that binds PKA. AKAP15 colocalizes with L-type calcium channels in transverse tubules and is associated with L-type calcium channels in transfected cells. A peptide fragment of AKAP15 encompassing the RII-binding domain blocks voltage-dependent potentiation. These results indicate that AKAP15 targets PKA to the calcium channel and plays a critical role in voltage-dependent potentiation and regulation of skeletal muscle contraction. The expression of AKAP15 in the brain and heart suggests that it may mediate rapid PKA regulation of L-type calcium channels in neurons and cardiac myocytes.

Detection and discrimination of PrPSc by multi-spectral ultraviolet fluorescence.

Prion diseases are progressive degenerative disorders of the central nervous system. The transmissibility and fatal nature of these diseases necessitate their rapid and accurate diagnosis. The hallmark of these diseases is the accumulation of PrPSc, a protease-resistant form of a host-coded glycoprotein. We have been evaluating the use of multi-spectral ultraviolet fluorescent spectroscopy as a means of detecting and distinguishing between different forms of PrPSc. Spectroscopic measurements of fluorescence from untreated and proteinase K (PK)-treated PrPSc, purified from 263K scrapie strain-infected hamster brains and ME7 scrapie strain-infected mouse brains, were performed. Spectra of untreated and PK-treated PrPSc samples for 263K and ME7 appeared qualitatively different. The identification and discrimination of PrPSc were possible based on these spectral signatures, calculations of their fluorescence cross sections, and determination of the orthogonal differences. This technique has the potential not only for the sensitive, specific, and direct detection of PrPSc, but also for the ability to distinguish between different forms of the prion protein.

Noninfective mitral valve vegetations identified by transesophageal echocardiography as a cause of stroke.

BACKGROUND: Transesophageal echocardiography (TEE) is a useful procedure to evaluate selected stroke patients for cardiac sources of embolism. To date, noninfective valvular vegetations have not been described in large studies using transesophageal echocardiography to detect cardiac sources of embolism. We sought to investigate the frequency of noninfective valvular vegetations in patients with unexplained stroke referred for TEE and to determine the relationship of these vegetations to unrecognized thrombophilic disorders. METHODS: We evaluated 641 consecutive patients referred for TEE as a result of unexplained stroke or transient ischemic attack for the presence of valvular vegetations. Of those with vegetations identified, serial blood cultures were obtained to evaluate for an infectious etiology. Patients also had serum testing for thrombophilic disorders and selected patients underwent cerebral angiography. RESULTS: Thirteen patients (2%) who underwent TEE evaluation for unexplained stroke or transient ischemic attack were found to have noninfective valvular vegetations, all involving the mitral valve; none were identified by transthoracic echocardiography. Antiphospholipid antibodies were identified in 8 of these 13 patients (62%) and a protein C deficiency in 1 patient (8%). CONCLUSIONS: Noninfective valvular vegetations are a potential cardiac source of embolism in patients with unexplained stroke that can be better identified using transesophageal echocardiography. A large percentage of these individuals have a previously unrecognized thrombophilic disorder.

Distinguishability of biological material by use of ultraviolet multispectral fluorescence.

Recent interest in the detection and analysis of biological samples by spectroscopic methods has led to questions concerning the degree of distinguishability and biological variability of the UV fluorescent spectra from such complex samples. We show that the degree of distinguishability of such spectra is readily determined numerically. As a practical example of this technique, we show its application to the analysis of UV fluorescence spectra taken of E. coli, S. aureus, and S. typhimurium. The use of this analysis to determine the degree of biological variability and also to verify that measurements are being made in a linear regime in which analytic methods such as multivariate analysis are valid is discussed.

Identification of a 15-kDa cAMP-dependent protein kinase-anchoring protein associated with skeletal muscle L-type calcium channels.

Voltage-dependent potentiation of skeletal muscle L-type calcium channels requires phosphorylation by cAMP-dependent protein kinase (PKA) that is localized by binding to a cAMP-dependent protein kinase-anchoring protein (AKAP). L-type calcium channels purified from rabbit skeletal muscle contain an endogenous co-purifying protein kinase activity that phosphorylates the alpha1 and beta subunits of the channel. The co-purifying kinase also phosphorylates a known PKA peptide substrate, is stimulated by cAMP, and is inhibited by PKA inhibitor peptide-(5-24), indicating that it is PKA. PKA activity co-immunoprecipitates with the calcium channel, suggesting that the channel and the kinase are physically associated. Using biotinylated type II regulatory subunit of PKA (RII) as a probe, we have identified a 15-kDa RII-binding protein in purified calcium channel preparations, which we have designated AKAP-15. Anti-peptide antibodies directed against the alpha1 subunit of the calcium channel co-immunoprecipitate AKAP-15. Together, these findings demonstrate a physical link between PKA and the calcium channel and suggest that AKAP-15 may mediate their interaction.