Non-Overlapping Functions for Pyk2 and FAK in Osteoblasts during Fluid Shear Stress-Induced Mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2+/+ and Pyk2−/− primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2+/+ and Pyk2−/− osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK−/− osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts

The imperative for controlled mechanical stresses in unraveling cellular mechanisms of mechanotransduction

BACKGROUND: In vitro mechanotransduction studies are designed to elucidate cell behavior in response to a well-defined mechanical signal that is imparted to cultured cells, e.g. through fluid flow. Typically, flow rates are calculated based on a parallel plate flow assumption, to achieve a targeted cellular shear stress. This study evaluates the performance of specific flow/perfusion chambers in imparting the targeted stress at the cellular level. METHODS: To evaluate how well actual flow chambers meet their target stresses (set for 1 and 10 dyn/cm(2 )for this study) at a cellular level, computational models were developed to calculate flow velocity components and imparted shear stresses for a given pressure gradient. Computational predictions were validated with micro-particle image velocimetry (μPIV) experiments. RESULTS: Based on these computational and experimental studies, as few as 66% of cells seeded along the midplane of commonly implemented flow/perfusion chambers are subjected to stresses within ±10% of the target stress. In addition, flow velocities and shear stresses imparted through fluid drag vary as a function of location within each chamber. Hence, not only a limited number of cells are exposed to target stress levels within each chamber, but also neighboring cells may experience different flow regimes. Finally, flow regimes are highly dependent on flow chamber geometry, resulting in significant variation in magnitudes and spatial distributions of stress between chambers. CONCLUSION: The results of this study challenge the basic premise of in vitro mechanotransduction studies, i.e. that a controlled flow regime is applied to impart a defined mechanical stimulus to cells. These results also underscore the fact that data from studies in which different chambers are utilized can not be compared, even if the target stress regimes are comparable

Understanding international volunteering: Who is most likely to participate?

International volunteers are playing an increasingly important role in meeting diverse challenges and improving lives of many across the world, and more opportunities for international volunteering are available across the nonprofit, government, and corporate sectors than ever. Nevertheless, little is known about international volunteering and the individuals who participate in this activity. This study examines the utility of the existing volunteering literature by testing how the traditional determinants of volunteering predict participation in international volunteering. The findings show that individuals who have attained post-secondary education, are from a high-income household, or are self-employed are most likely to volunteer internationally. The results also indicate that some of the known predictors of volunteering, such as age, race and gender, do not satisfactorily explain participation in international volunteering. This study concludes by providing suggestions for recruitment strategies, calling for further research on international volunteering

The Shedding of CD62L (L-Selectin) Regulates the Acquisition of Lytic Activity in Human Tumor Reactive T Lymphocytes

CD62L/L-selectin is a marker found on naïve T cells and further distinguishes central memory (Tcm, CD62L+) from effector memory (Tem, CD62L−) T cells. The regulation of CD62L plays a pivotal role in controlling the traffic of T lymphocytes to and from peripheral lymph nodes. CD62L is shed from the cell membrane following T cell activation, however, the physiological significance of this event remains to be elucidated. In this study, we utilized in vitro generated anti-tumor antigen T cells and melanoma lines as a model to evaluate the dynamics of CD62L shedding and expression of CD107a as a marker of lytic activity. Upon encounter, with matched tumor lines, antigen reactive T cells rapidly lose CD62L expression and this was associated with the acquisition of CD107a. By CD62L ELISA, we confirmed that this transition was mediated by the shedding of CD62L when T cells encountered specific tumor antigen. The introduction of a shedding resistant mutant of CD62L into the tumor antigen-reactive T cell line JKF6 impaired CD107a acquisition following antigen recognition and this was correlated with decreased lytic activity as measured by 51Cr release assays. The linkage of the shedding of CD62L from the surface of anti-tumor T cells and acquisition of lytic activity, suggests a new function for CD62L in T cell effector functions and anti-tumor activity

Dystroglycan versatility in cell adhesion: a tale of multiple motifs

Dystroglycan is a ubiquitously expressed heterodimeric adhesion receptor. The extracellular a-subunit makes connections with a number of laminin G domain ligands including laminins, agrin and perlecan in the extracellular matrix and the transmembrane b-subunit makes connections to the actin filament network via cytoskeletal linkers including dystrophin, utrophin, ezrin and plectin, depending on context. Originally discovered as part of the dystrophin glycoprotein complex of skeletal muscle, dystroglycan is an important adhesion molecule and signalling scaffold in a multitude of cell types and tissues and is involved in several diseases. Dystroglycan has emerged as a multifunctional adhesion platform with many interacting partners associating with its short unstructured cytoplasmic domain. Two particular hotspots are the cytoplasmic juxtamembrane region and at the very carboxy terminus of dystroglycan. Regions which between them have several overlapping functions: in the juxtamembrane region; a nuclear localisation signal, ezrin/radixin/moesin protein, rapsyn and ERK MAP Kinase binding function, and at the C terminus a regulatory tyrosine governing WW, SH2 and SH3 domain interactions. We will discuss the binding partners for these motifs and how their interactions and regulation can modulate the involvement of dystroglycan in a range of different adhesion structures and functions depending on context. Thus dystroglycan presents as a multifunctional scaffold involved in adhesion and adhesion-mediated signalling with its functions under exquisite spatiotemporal regulation

The emergence of health inequalities in early adulthood: evidence on timing and mechanisms from a West of Scotland cohort

Background Evidence is inconsistent as to whether or not there are health inequalities in adolescence according to socio-economic position (SEP) and whether or when they emerge in early adulthood. Despite the large health inequalities literature, few studies have simultaneously compared the relative importance of ?health selection? versus ?social causation? at this life-stage. This study followed a cohort through the youth-adult transition to: (1) determine whether, and if so, when, health inequalities became evident according to both class of origin and current SEP; (2) compare the importance of health selection and social causation mechanisms; and (3) investigate whether these phenomena vary by gender. Methods Data are from a West-of-Scotland cohort, surveyed five times between age 15 (in 1987, N=1,515, response=85%) and 36. Self-reported physical and mental health were obtained at each survey. SEP was based on parental occupational class at 15, a combination of own education or occupational status at 18 and own occupational class (with an additional non-employment category) at older ages. In respect of when inequalities emerged, we used the relative index of inequality to examine associations between both parental and own current SEP and health at each age. In respect of mechanisms, path models, including SEP and health at each age, investigated both inter and intra-generational paths from SEP to health (?causation?) and from health to SEP (?selection?). Analyses were conducted separately for physical and mental health, and stratified by gender. Results Associations between both physical and mental health and parental SEP were non-significant at every age. Inequalities according to own SEP emerged for physical health at 24 and for mental health at 30. There was no evidence of selection based on physical health, but some evidence of associations between mental health in early adulthood and later SEP (intra-generational selection). Paths indicated intra-generational (males) and inter-generational (females) social causation of physical health inequalities, and intra-generational (males and females) and inter-generational (females) social causation of mental health inequalities. Conclusions The results suggest complex and reciprocal relationships between SEP and health and highlight adolescence and early adulthood as a sensitive period for this process, impacting on future life-chances and health

Racism as a determinant of health: a systematic review and meta-analysis

Despite a growing body of epidemiological evidence in recent years documenting the health impacts of racism, the cumulative evidence base has yet to be synthesized in a comprehensive meta-analysis focused specifically on racism as a determinant of health. This meta-analysis reviewed the literature focusing on the relationship between reported racism and mental and physical health outcomes. Data from 293 studies reported in 333 articles published between 1983 and 2013, and conducted predominately in the U.S., were analysed using random effects models and mean weighted effect sizes. Racism was associated with poorer mental health (negative mental health: r = -.23, 95% CI [-.24,-.21], k = 227; positive mental health: r = -.13, 95% CI [-.16,-.10], k = 113), including depression, anxiety, psychological stress and various other outcomes. Racism was also associated with poorer general health (r = -.13 (95% CI [-.18,-.09], k = 30), and poorer physical health (r = -.09, 95% CI [-.12,-.06], k = 50). Moderation effects were found for some outcomes with regard to study and exposure characteristics. Effect sizes of racism on mental health were stronger in cross-sectional compared with longitudinal data and in non-representative samples compared with representative samples. Age, sex, birthplace and education level did not moderate the effects of racism on health. Ethnicity significantly moderated the effect of racism on negative mental health and physical health: the association between racism and negative mental health was significantly stronger for Asian American and Latino(a) American participants compared with African American participants, and the association between racism and physical health was significantly stronger for Latino(a) American participants compared with African American participants.<br /