Prx1-Expressing Progenitor Primary Cilia Mediate Bone Formation in response to Mechanical Loading in Mice.

Increases in mechanical loading can enhance the addition of new bone, altering geometry and density such that bones better withstand higher forces. Bone-forming osteoblasts have long been thought to originate from progenitors, but the exact source is yet to be identified. Previous studies indicate osteogenic precursors arise from Prx1-expressing progenitors during embryonic development and adult fracture repair. However, it is unknown whether this cell population is also a source for mechanically induced active osteoblasts. We first identified that Prx1 is expressed in skeletally mature mouse periosteum, a thin tissue covering the surface of the bone that is rich in osteoprogenitors. We then traced Prx1 progenitor lineage using a transgenic mouse model carrying both a Prx1-driven tamoxifen-inducible Cre and a ROSA-driven lacZ reporter gene. Cells that expressed Prx1 when compressive axial loading was applied were detected within the cortical bone days after stimulation, indicating osteocytes are of Prx1-expressing cell origin. In addition, we evaluated how these cells sense and respond to physical stimulation in vivo by disrupting their primary cilia, which are antenna-like sensory organelles known to enhance mechanical and chemical signaling kinetics. Although Prx1-driven primary cilium disruption did not affect osteoblast recruitment to the bone surface, the relative mineral apposition and bone formation rates were decreased by 53% and 34%, respectively. Thus, this cell population contributes to load-induced bone formation, and primary cilia are needed for a complete response. Interestingly, Prx1-expressing progenitors are easily extracted from periosteum and are perhaps an attractive alternative to marrow stem cells for bone tissue regeneration strategies.

Association of Husband Smoking With Wife's Hypertension Status in Over 5 Million Chinese Females Aged 20 to 49 Years.

BACKGROUND: The effect of second-hand smoking, especially husband smoking, on wife's hypertension has not been well studied. The current study was aimed to assess the association of husband smoking with wife's hypertension among females aged 20 to 49 years. METHODS AND RESULTS: This study included 5 027 731 females along with their husbands from the National Free Pre-pregnancy Checkup Projects conducted across 31 provinces in China in 2014. Smoking/passive smoking status was collected by a standard questionnaire and blood pressure was measured by an electronic device after 10 minutes rest. Odds ratios and their corresponding 95% CIs for female hypertension were estimated according to smoking status of husband and wife, husbands' smoking amount, and cumulative exposure to husband smoking. Compared with neither-smoker group, the multivariable-adjusted odds ratio for female hypertension was 1.28 (1.27-1.30), 1.53 (1.30-1.79), and 1.50 (1.36-1.67) in husband-only, wife-only, and mixed group, respectively. Furthermore, a higher risk of having hypertension was associated with amount and cumulative exposure of husband smoking. For example, compared with neither-smoker, the multivariate-adjusted odds ratio was 1.22 (1.19-1.25), 1.24 (1.21-1.26), 1.32 (1.26-1.37), 1.37 (1.34-1.41), and 1.75 (1.64-1.87) for females whose husband smoked 1 to 5, 6 to 10, 11 to 15, 16 to 20, and ≥21 cigarettes per day, respectively (Pfor trend<0.001). Subgroup analyses identified similar results. CONCLUSIONS: There were associations of husband smoking with female hypertension prevalence. A family-based smoking restriction strategy may reduce smoking in males and improve hypertension control in females.

Is Colorectal Cancer A Western Disease? Role of Knowledge and Influence of Misconception on Colorectal Cancer Screening among Chinese and Korean Americans: A Mixed Methods Study

Background: Chinese and Korean Americans have lower colorectal cancer (CRC) screening rates than other racial/ ethnic groups, which may be explained by a low level of CRC knowledge and a high level of misconceptions. This study explores the role of knowledge in CRC screening among these groups. Methods: Chinese (N=59) and Korean (N=61) Americans older than 50 were recruited from the Washington D.C. Metropolitan area. They completed a detailed survey and participated in focus groups to discuss their knowledge on CRC and CRC screening. Seventeen physicians, community leaders, and patient navigators participated in key informant interviews. Using a mixed methods approach, data were analyzed quantitatively and qualitatively. Results: Participants lacked knowledge about CRC and CRC screening. More than half did not know that screening begins at age 50 and there are several types of tests available. More than 30% thought CRC screening was not necessary if there were no symptoms or there was nothing they could do to prevent CRC. Focus group findings suggested understanding about CRC was limited by an inadequate source of linguistically and culturally relevant health information. For example, many participants considered CRC a western condition mainly caused by unhealthy diet. This led to under-estimations about their susceptibility to CRC. Knowledge was positively associated with self-reported screening. Participants who had higher knowledge scores were more likely to report ever having had a colonoscopy and confidence in ability to have CRC screening. Conclusions: Mixed-methods analysis provides multi-faceted perspectives on CRC knowledge and its influence on screening. Study findings can help inform interventions to increase CRC screening among Chinese and Korean Americans.

Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism.

It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitmentin vitro In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cellsin vivo Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitmentin vivoand that the primary cilium contributes to this process.-Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism.

Epigenetic changes during mechanically induced osteogenic lineage commitment.

Osteogenic lineage commitment is often evaluated by analyzing gene expression. However, many genes are transiently expressed during differentiation. The availability of genes for expression is influenced by epigenetic state, which affects the heterochromatin structure. DNA methylation, a form of epigenetic regulation, is stable and heritable. Therefore, analyzing methylation status may be less temporally dependent and more informative for evaluating lineage commitment. Here we analyzed the effect of mechanical stimulation on osteogenic differentiation by applying fluid shear stress for 24 hr to osteocytes and then applying the osteocyte-conditioned medium (CM) to progenitor cells. We analyzed gene expression and changes in DNA methylation after 24 hr of exposure to the CM using quantitative real-time polymerase chain reaction and bisulfite sequencing. With fluid shear stress stimulation, methylation decreased for both adipogenic and osteogenic markers, which typically increases availability of genes for expression. After only 24 hr of exposure to CM, we also observed increases in expression of later osteogenic markers that are typically observed to increase after seven days or more with biochemical induction. However, we observed a decrease or no change in early osteogenic markers and decreases in adipogenic gene expression. Treatment of a demethylating agent produced an increase in all genes. The results indicate that fluid shear stress stimulation rapidly promotes the availability of genes for expression, but also specifically increases gene expression of later osteogenic markers.

Mechanically induced osteogenic lineage commitment of stem cells.

Bones adapt to accommodate the physical forces they experience through changes in architecture and mass. Stem cells differentiate into bone-forming osteoblasts, and mechanical stimulation is involved in this process. Various studies have applied controlled mechanical stimulation to stem cells and investigated the effects on osteogenic lineage commitment. These studies demonstrate that physical stimuli can induce osteogenic lineage commitment. Tension, fluid shear stress, substrate material properties, and cell shape are all factors that influence osteogenic differentiation. In particular, the level of tension is important. Also, rigid substrates with stiffness similar to collagenous bone induce osteogenic differentiation, while softer substrates induce other lineages. Finally, cells allowed to adhere over a larger area are able to differentiate towards the osteogenic lineage while cells adhering to a smaller area are restricted to the adipogenic lineage. Stem cells are able to sense their mechanical environments through various mechanosensors, including the cytoskeleton, focal adhesions, and primary cilia. The cytoskeleton provides a structural frame for the cell, and myosin interacts with actin to generate cytoskeletal tension, which is important for mechanically induced osteogenesis of stem cells. Adapter proteins link the cytoskeleton to integrins, which attach the cell to the substrate, forming a focal adhesion. A variety of signaling proteins are also associated with focal adhesions. Forces are transmitted to the substrate at these sites, and an intact focal adhesion is important for mechanically induced osteogenesis. The primary cilium is a single, immotile, antenna-like structure that extends from the cell into the extracellular space. It has emerged as an important signaling center, acting as a microdomain to facilitate biochemical signaling. Mechanotransduction is the process by which physical stimuli are converted into biochemical responses. When potential mechanosensors are disrupted, the activities of components of mechanotransduction pathways are also inhibited, preventing mechanically induced osteogenesis. Calcium, mitogen-activated protein kinase/extracellular signal-regulated kinase, Wnt, Yes-associated protein/transcriptional coactivator with PDZ-binding motif and RhoA/Rho kinase signaling are some of the mechanotransduction pathways proposed to be important. In this review, types of mechanical stimuli, mechanosensors, and key pathways involved in mechanically induced osteogenesis of stem cells are discussed.

Focal adhesion kinase plays a role in osteoblast mechanotransduction in vitro but does not affect load-induced bone formation in vivo.

A healthy skeleton relies on bone's ability to respond to external mechanical forces. The molecular mechanisms by which bone cells sense and convert mechanical stimuli into biochemical signals, a process known as mechanotransduction, are unclear. Focal adhesions play a critical role in cell survival, migration and sensing physical force. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that controls focal adhesion dynamics and can mediate reparative bone formation in vivo and osteoblast mechanotransduction in vitro. Based on these data, we hypothesized that FAK plays a role in load-induced bone formation. To test this hypothesis, we performed in vitro fluid flow experiments and in vivo bone loading studies in FAK-/- clonal lines and conditional FAK knockout mice, respectively. FAK-/- osteoblasts showed an ablated prostaglandin E(2) (PGE(2)) response to fluid flow shear. This effect was reversed with the re-expression of wild-type FAK. Re-expression of FAK containing site-specific mutations at Tyr-397 and Tyr-925 phosphorylation sites did not rescue the phenotype, suggesting that these sites are important in osteoblast mechanotransduction. Interestingly, mice in which FAK was conditionally deleted in osteoblasts and osteocytes did not exhibit altered load-induced periosteal bone formation. Together these data suggest that although FAK is important in mechanically-induced signaling in osteoblasts in vitro, it is not required for an adaptive response in vivo, possibly due to a compensatory mechanism that does not exist in the cell culture system.

The primary cilium as a novel extracellular sensor in bone.

Mechanically induced adaptation of bone is required to maintain a healthy skeleton and defects in this process can lead to dramatic changes in bone mass, resulting in bone diseases such as osteoporosis. Therefore, understanding how this process occurs could yield novel therapeutics to treat diseases of excessive bone loss or formation. Over the past decade the primary cilium has emerged as a novel extracellular sensor in bone, being required to transduce changes in the extracellular mechanical environment into biochemical responses regulating bone adaptation. In this review, we introduce the primary cilium as a novel extracellular sensor in bone; discuss the in vitro and in vivo findings of primary cilia based sensing in bone; explore the role of the primary cilium in regulating stem cell osteogenic fate commitment and finish with future directions of research and possible development of cilia targeting therapeutics to treat bone diseases.