The ability of exercise to decrease fat mass and increase bone mass may occur through mechanical biasing of mesenchymal stem cells (MSCs) away from adipogenesis and toward osteoblastogenesis. C3H10T1/2 MSCs cultured in highly adipogenic medium express peroxisome proliferator-activated receptor gamma and adiponectin mRNA and protein, and accumulate intracellular lipid. Mechanical strain applied for 6 h daily inhibited expression of peroxisome proliferator-activated receptor gamma and adiponectin mRNA by up to 35 and 50%, respectively, after 5 d. A decrease in active and total beta-catenin levels during adipogenic differentiation was entirely prevented by daily application of mechanical strain; furthermore, strain induced beta-catenin nuclear translocation. Inhibition of glycogen synthase kinase-3beta by lithium chloride or SB415286 also prevented adipogenesis, suggesting that preservation of beta-catenin levels was important to strain inhibition of adipogenesis. Indeed, mechanical strain inactivated glycogen synthase kinase-3beta, which was preceded by Akt activation, indicating that strain transmits antiadipogenic signals through this pathway. Cells grown under adipogenic conditions showed no increase in osteogenic markers runt-related transcription factor (Runx) 2 and osterix (Osx); subsequent addition of bone morphogenetic protein 2 for 2 d increased Runx2 but not Osx expression in unstrained cultures. When cultures were strained for 5 d before bone morphogenetic protein 2 addition, Runx2 mRNA increased more than in unstrained cultures, and Osx expression more than doubled. As such, mechanical strain enhanced MSC potential to enter the osteoblast lineage despite exposure to adipogenic conditions. Our results indicate that MSC commitment to adipogenesis can be suppressed by mechanical signals, allowing other signals to promote osteoblastogenesis. These data suggest that positive effects of exercise on both fat and bone may occur during mesenchymal lineage selection.

. Modulation of membrane channel currents by gap junction protein mimetic peptides: size matters. Am J Physiol Cell Physiol 293: C1112-C1119, 2007. First published July 27, 2007; doi:10.1152/ajpcell.00097.2007.-Connexin mimetic peptides are widely used to assess the contribution of nonjunctional connexin channels in several processes, including ATP release. These peptides are derived from various connexin sequences and have been shown to attenuate processes downstream of the putative channel activity. Yet so far, no documentation of effects of peptides on connexin channels has been presented. We tested several connexin and pannexin mimetic peptides and observed attenuation of channel currents that is not compatible with sequence specific actions of the peptides. Connexin mimetic peptides inhibited pannexin channel currents but not the currents of the channel formed by connexins from which the sequence was derived. Pannexin mimetic peptides did inhibit pannexin channel currents but also the channels formed by connexin 46. The same pattern of effects was observed for dye transfer, except that the inhibition levels were more pronounced than for the currents. The channel inhibition by peptides shares commonalities with channel effects of polyethylene glycol (PEG), suggesting a steric block as a mechanism. PEG accessibility is in the size range expected for the pore of innexin gap junction channels, consistent with a functional relatedness of innexin and pannexin channels.

Mechanical loading of bone initiates an anabolic, anticatabolic pattern of response, yet the molecular events involved in mechanical signal transduction are not well understood. Wnt/ ␤-catenin signaling has been recognized in promoting bone anabolism, and application of strain has been shown to induce ␤-catenin activation. In this work, we have used a preosteoblastic cell line to study the effects of dynamic mechanical strain on ␤-catenin signaling. We found that mechanical strain caused a rapid, transient accumulation of active ␤-catenin in the cytoplasm and its translocation to the nucleus. This was followed by up-regulation of the Wnt/␤-catenin target genes Wisp1 and Cox2, with peak responses at 4 and 1 h of strain, respectively. The increase of ␤-catenin was temporally related to the activation of Akt and subsequent inactivation of GSK3␤, and caveolin-1 was not required for these molecular events. Application of Dkk-1, which disrupts canonical Wnt/LRP5 signaling, did not block strain-induced nuclear translocation of ␤-catenin or upregulation of Wisp1 and Cox2 expression. Conditions that increased basal ␤-catenin levels, such as lithium chloride treatment or repression of caveolin-1 expression, were shown to enhance the effects of strain. In summary, mechanical strain activates Akt and inactivates GSK3␤ to allow ␤-catenin translocation, and Wnt signaling through LRP5 is not required for these strain-mediated responses. Thus, ␤-catenin serves as both a modulator and effector of mechanical signals in bone cells.Bone tissue undergoes remodeling throughout life to adapt to its mechanical load, resulting in a mass that is optimized for daily mechanical demands. The remodeling process is tightly regulated by a balance between the number of bone-producing osteoblasts and bone-resorbing osteoclasts. Application of mechanical load promotes bone formation, whereas the removal of load leads to bone loss (1, 2). More recently, an important role for Wnt/␤-catenin signaling has been recognized in promoting bone anabolism (3), and interestingly, mice with a loss-of-function mutation in the Wnt co-receptor LRP5 were shown to be resistant to the positive effects of local bone loading (4). Information further linking mechanically induced bone formation with activation of Wnt/␤-catenin processes has emerged with evidence that straining cells causes translocation of ␤-catenin into the cell nucleus (5, 6). Consistent with ␤-catenin activation, osteoblasts respond to mechanical loading with increased expression of Wnt/␤-catenin target genes, including Sfrp1 (secreted frizzled-related protein 1) and cyclin D1 (5).The loss of ␤-catenin has been found to disrupt both skeletal development and postnatal bone acquisition (7,8), establishing the importance of ␤-catenin signaling in osteoblast differentiation and function. ␤-Catenin can be found in at least two different cellular pools, suggesting compartmentalized roles. The pool of ␤-catenin found at the plasma membrane is bound to E-cadherin and ␣-catenin in adherens junctions. The sol...

Gap junction channels are intercellular channels that mediate the gated transfer of molecules between adjacent cells. To identify the domain determining channel conductance, the first transmembrane segment (M1) was reciprocally exchanged between Cx46 and Cx32E(1)43. The resulting chimeras exhibited conductances similar to that of the respective M1 donor. Furthermore, a chimera with the carboxy-terminal half of M1 in Cx46 replaced by that of Cx32 exhibited a conductance similar to that of Cx32E(1)43, whereas the chimera with only the amino-terminal half of M1 replaced retained the unitary conductance of wild-type Cx46. Extending the M1 domain swapping to other connexins by replacing the carboxy-terminal half of M1 in Cx46 with that of Cx37 yielded a chimera channel with increased unitary conductance close to that of Cx37. Furthermore, a point mutant of Cx46, with leucine substituted by glycine in position 35, displayed a conductance much larger than that of the wild type. Thus, the M1 segment, especially the second half, contains important determinants of conductance of the connexin channel.

The physiological function of gap junction channels goes well beyond their initially discovered role in electrical synchronization of excitable cells. In most tissues, gap junction cells facilitate the exchange of second messengers and metabolites between cells. To test which parts of the channels formed by connexins determine the exclusion limit for the transit of molecules in the size range of second messengers and metabolites a domain exchange approach was used in combination with an accessibility assay for nonelectrolytes and flux measurements. The experimental results suggest that two open hemichannel forming connexins, Cx46 and Cx32E(1)43, differ in accessibility and permeability. Sucrose is at the exclusion limit for Cx46 channels whereas sorbitol is at the exclusion limit for Cx32E(1)43 channels. In chimeras between these connexins, where the first transmembrane segment M1 is exchanged, the exclusion limits correlate with those of the M1 donor. The same segregation was found in a separate study for the unitary conductance of the channels. Thus, conductance and permeability/accessibility of the channels cosegregate with M1.

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