Francis J. Alenghat

Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca2+ influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP3 receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

Mechanotransduction: All Signals Point to Cytoskeleton, Matrix, and Integrins

Mechanical stresses modulate cell function by either activating or tuning signal transduction pathways. Mechanotransduction, the process by which cells convert mechanical stimuli into a chemical response, occurs both in cells specialized for sensing mechanical cues and in parenchymal cells whose primary function is not mechanosensory. However, common among the various responses to mechanical stress is the importance of direct or indirect connections between the internal cytoskeleton, the extracellular matrix (ECM), and traditional signal transducing molecules. In many instances, these elements converge at focal adhesions, sites of structural attachment between the cytoskeleton and ECM that are anchored by cell surface integrin receptors. Alenghat and Ingber discuss the accumulating evidence for the central role of cytoskeleton, ECM, and integrin-anchored focal adhesions in several mechanotransduction pathways.

Mechanical control of cyclic AMP signalling and gene transcription through integrins

This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.

Mechanical control of cAMP signaling through integrins is mediated by the heterotrimeric Gαs protein

Mechanical stresses that are preferentially transmitted across the cell surface via transmembrane integrin receptors activate gene transcription by triggering production of intracellular chemical second messengers, such as cAMP. Here we show that the sensitivity of the cAMP signaling pathway to mechanical stresses transferred across β1 integrins is mediated by force‐dependent activation of the heterotrimeric G protein subunit Gαs within focal adhesions at the site of stress application. Gαs is recruited to focal adhesions that form within minutes following clustering of β1 integrins induced by cell binding to magnetic microbeads coated with activating integrin ligands, and β1 integrin and Gαs co‐precipitate when analyzed biochemically. Stress application to activated β1 integrins using magnetic twisting cytometry increases Gαs recruitment and activates these large G proteins within focal adhesions, as measured by binding of biotinylated azido‐anilido‐GTP, whereas application of similar stresses to inactivated integrins or control histocompatibility antigens has little effect. This response is relevant physiologically as application of mechanical strain to cells bound to flexible extracellular matrix‐coated substrates induce translocation of phospho‐CREB to the nucleus, which can be attenuated by inhibiting Gαs activity, either using the inhibitor melittin or suppressing its expression using siRNA. Although integrins are not typical G protein‐coupled receptors, these results show that integrins focus mechanical stresses locally on heterotrimeric G proteins within focal adhesions at the site of force application, and transduce mechanical stimuli into an intracellular cAMP signaling response by activating Gαs at these membrane signaling sites. J. Cell. Biochem. 106: 529–538, 2009.

Macrophages require Skap2 and Sirpα for integrin-stimulated cytoskeletal rearrangement

Summary Macrophages migrate to sites of insult during normal inflammatory responses. Integrins guide such migration, but the transmission of signals from integrins into the requisite cytoskeletal changes is poorly understood. We have discovered that the hematopoietic adaptor protein Skap2 is necessary for macrophage migration, chemotaxis, global actin reorganization and local actin reorganization upon integrin engagement. Binding of phosphatidylinositol [3,4,5]-triphosphate to the Skap2 pleckstrin-homology (PH) domain, which relieves its conformational auto-inhibition, is critical for this integrin-driven cytoskeletal response. Skap2 enables integrin-induced tyrosyl phosphorylation of Src-family kinases (SFKs), Adap, and Sirp&agr;, establishing their roles as signaling partners in this process. Furthermore, macrophages lacking functional Sirp&agr; unexpectedly have impaired local integrin-induced responses identical to those of Skap2−/− macrophages, and Skap2 requires Sirp&agr; for its recruitment to engaged integrins and for coordinating downstream actin rearrangement. By revealing the positive-regulatory role of Sirp&agr; in a Skap2-mediated mechanism connecting integrin engagement with cytoskeletal rearrangement, these data demonstrate that Sirp&agr; is not exclusively immunoinhibitory, and illuminate previously unexplained observations implicating Skap2 and Sirp&agr; in mouse models of inflammatory disease.

Membrane Protein Dynamics and Functional Implications in Mammalian Cells

The organization of the plasma membrane is both highly complex and highly dynamic. One manifestation of this dynamic complexity is the lateral mobility of proteins within the plane of the membrane, which is often an important determinant of intermolecular protein-binding interactions, downstream signal transduction, and local membrane mechanics. The mode of membrane protein mobility can range from random Brownian motion to immobility and from confined or restricted motion to actively directed motion. Several methods can be used to distinguish among the various modes of protein mobility, including fluorescence recovery after photobleaching, single-particle tracking, fluorescence correlation spectroscopy, and variations of these techniques. Here, we present both a brief overview of these methods and examples of their use to elucidate the dynamics of membrane proteins in mammalian cells-first in erythrocytes, then in erythroblasts and other cells in the hematopoietic lineage, and finally in non-hematopoietic cells. This multisystem analysis shows that the cytoskeleton frequently governs modes of membrane protein motion by stably anchoring the proteins through direct-binding interactions, by restricting protein diffusion through steric interactions, or by facilitating directed protein motion. Together, these studies have begun to delineate mechanisms by which membrane protein dynamics influence signaling sequelae and membrane mechanical properties, which, in turn, govern cell function.

The Prevalence of Atherosclerosis in Those with Inflammatory Connective Tissue Disease by Race, Age, and Traditional Risk Factors

Systemic inflammation promotes cardiovascular disease. Inflammatory connective tissue diseases (CTD) like lupus and rheumatoid arthritis associate with cardiovascular risk, but it is unknown whether particular groups of patients have enhanced propensity for atherosclerotic cardiovascular disease (ASCVD) associated with their CTD. Analysis of aggregate health record data at a large U.S. academic center identified CTD and ASCVD status for 287,467 African American and white adults. ASCVD prevalence in those with CTD was 29.7% for African Americans and 14.7% for white patients with prevalence ratios, compared to those without CTD, of 3.1 and 1.8, respectively. When different types of CTD were analyzed individually (rheumatoid arthritis; lupus; scleroderma; Sjögren Syndrome; dermatomyositis/polymyositis; unspecified/mixed CTD; other inflammatory arthropathy), increased ASCVD rates were found in nearly all subsets, always with higher prevalence ratios in African Americans. The prevalence ratio of ASCVD was particularly high in young African Americans. Furthermore, individuals lacking traditional cardiovascular risk factors had more ASCVD if they had CTD (prevalence ratio 2.9). Multivariate analysis confirmed a positive interaction between CTD and African-American race and a negative interaction between CTD and age. The factors driving the observed disproportionate CTD-associated ASCVD in African Americans, young adults, and those without traditional risk factors warrant further study.

Protein Mimetic and Anticancer Properties of Monocyte-Targeting Peptide Amphiphile Micelles

Monocyte chemoattractant protein-1 (MCP-1) stimulates the migration of monocytes to inflammatory sites, leading to the progression of many diseases. Recently, we described a monocyte-targeting peptide amphiphile micelle (MCP-1 PAM) incorporated with the chemokine receptor CCR2 binding motif of MCP-1, which has a high affinity for monocytes in atherosclerotic plaques. We further report here the biomimetic components of MCP-1 PAMs and the influence of the nanoparticle upon binding to monocytes. We report that MCP-1 PAMs have enhanced secondary structure compared to the MCP-1 peptide. As a result, MCP-1 PAMs displayed improved binding and chemoattractant properties to monocytes, which upregulated the inflammatory signaling pathways responsible for monocyte migration. Interestingly, when MCP-1 PAMs were incubated in the presence of prostate cancer cells in vitro, the particle displayed anticancer efficacy by reducing CCR2 expression. Given that monocytes play an important role in tumor cell migration and invasion, our results demonstrate that PAMs can improve the native biofunctional properties of the peptide and may be used as an effective inhibitor to prevent chemokine-receptor interactions that promote disease progression.

Giant left ventricular aneurysm as a late complication of inferior myocardial infarction

After 3 days of nausea and dyspnoea, a 62-year-old man was found to have a completed inferior myocardial infarction. Angiography demonstrated right coronary artery occlusion, and no intervention was performed. Several days later, he had worsened dyspnoea and syncope due to pericardial haematoma. This prompted emergent surgical evacuation and patch repair of a 2-cm inferior wall rupture. Three months later, he presented …

Reverse Engineered Virtual Patient Populations as Surrogates for Real Patient-Level Data

Dissemination of clinical data for research has limitations. The most coveted data is richly descriptive at the individual level, but acquiring such granularity comes with significant cost, effort, or time. De-identification of individual records is not foolproof, with potential for privacy breaches, especially for “real-world” data derived from electronic health records. Also, the open data movement has progressed slowly for clinical trials, partly due to concerns about data ownership. Here, reverse engineered virtual patient populations (RE-ViPPs) are described, based on aggregate cross-tabulated categorical data from populations. The method does not require end-user access to individual-level data. Rather, using sequential linear regressions and random number generation, it generates virtual individual patients to comprise populations that, on average, closely resemble the real population in question. The method is validated by applying it to aggregated data derived from the seminal SPRINT trial, for which the individual-level data is known. The method yields virtual populations, each with 9361 patients, faithfully mimicking the 9361 real SPRINT participants. Multiple logistic regression on 100 such populations shows that, just as in SPRINT, risk factors with the highest odds ratio for the primary event are, in descending order, past clinical cardiovascular disease, age ≥ 75, chronic kidney disease, high non-HDL, and smoking history. Factors associated with fewer events are female sex and intensive blood pressure treatment (the trial’s intervention). Application of RE-ViPPs to trials, registries, and health record databases could reduce the cost, time, ownership, and de-identification burdens hindering open data by encouraging dissemination of aggregate, richly cross-tabulated real data that investigators can use to construct virtual patients and make meaningful conclusions.