Browsing by Author "Harris, Thurl E."

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    Mechanism of sphingosine 1-phosphate clearance from blood
    Kharel, Yugesh; Huang, Tao; Salamon, Anita; Harris, Thurl E.; Santos, Webster L.; Lynch, Kevin R. (Portland Press, 2020-03-06)
    The interplay of sphingosine 1-phosphate (S1P) synthetic and degradative enzymes as well as S1P exporters creates concentration gradients that are a fundamental to S1P biology. Extracellular S1P levels, such as in blood and lymph, are high relative to cellular S1P. The blood-tissue S1P gradient maintains endothelial integrity while local S1P gradients influence immune cell positioning. Indeed, the importance of S1P gradients was recognized initially when the mechanism of action of an S1P receptor agonist used as a medicine for multiple sclerosis was revealed to be inhibition of T-lymphocytes’ recognition of the high S1P in efferent lymph. Furthermore, the increase in erythrocyte S1P in response to hypoxia influences oxygen delivery during high altitude acclimatization. However, understanding of how S1P gradients are maintained is incomplete. For example, S1P is synthesized but is only slowly metabolized by blood yet circulating S1P turns over quickly by an unknown mechanism. Prompted by the counterintuitive observation that blood S1P increases markedly in response to inhibition S1P synthesis (by sphingosine kinase 2 (SphK2)), we studied mice wherein several tissues were made deficient in either SphK2 or S1P degrading enzymes. Our data reveal a mechanism whereby S1P is de-phosphorylated at the hepatocyte surface and the resulting sphingosine is sequestered by SphK phosphorylation and in turn degraded by intracellular S1P lyase. Thus, we identify the liver as the primary site of blood S1P clearance and provide an explanation for the role of SphK2 in this process. Our discovery suggests a general mechanism whereby S1P gradients are shaped.
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    Scleraxis-mediated regulation of tendon and ligament cell mechanobiology
    Nichols, Anne Elizabeth Carmack (Virginia Tech, 2018-06-12)
    Tendon and ligament injuries are common orthopedic problems that have an enormous impact on the quality of life of affected patients. Despite the frequency at which these injuries occur, current treatments are unable to restore native function to the damaged tissue. Because of this, reinjury is common. It is well known that mechanical stimulation is beneficial for promoting tendon and ligament development and tissue homeostasis; however, the specific mechanisms remain unclear. The transcription factor scleraxis (Scx) is an interesting candidate for mediating the tendon and ligament mechanoresponse, as it has been shown that Scx expression is induced by cyclic mechanical strain in tenocytes and is required for mechanically-induced stem cell tenogenesis. Moreover, Scx expression is increased in adult tendons following exercise. The studies described in this dissertation therefore focus on the combined role of Scx and mechanical stimulation in two contexts: 1) influencing ligament cell differentiation and 2) regulating adult tenocyte behavior. In the first study, transient Scx overexpression combined with mechanical strain in a 3D collagen hydrogel model was investigated as a means of deriving mature ligament cells from stem cells for use in ligament tissue engineering. Scx overexpression in C3H10T1/2 cells cultured in collagen hydrogels under static strain resulted in increased construct contraction and cell elongation, but no concurrent increase in the expression of ligament-related genes or production of glycosaminoglycans (GAG). When combined with low levels of cyclic strain, Scx overexpression resulted in increased mechanical properties of the tissue constructs, increased GAG production, and increased expression of ligament-related genes compared to cyclic strain alone. Together, these results demonstrate that Scx overexpression combined with cyclic strain can induce ligament cell differentiation and suggest that Scx does so by improving the mechanosensitivity of cells to cyclic strain. In the second study, the role of Scx in adult tenocyte mechanotransduction was explored using RNA-sequencing (RNA-seq) and small interfering RNA (siRNA) technologies. Equine tenocytes were exposed to siRNA targeting Scx or a control siRNA and maintained under cyclic mechanical strain prior to being submitted for RNA-seq. Comparison of the resulting transcriptomes revealed that Scx knockdown decreased the expression of several genes encoding important focal adhesion adaptor proteins. Correspondingly, Scx-depleted tenocytes showed abnormally long focal adhesions, decreased cytoskeletal stiffness, and an impaired ability to migrate on soft surfaces. This suggests that Scx regulates the tenocyte mechanoresponse by promoting the expression of focal adhesion-related genes. Combined, the results of these studies support a role for Scx in tendon and ligament cell mechanotransduction and identify the regulation of genes related to maintaining the cell-extracellular matrix connection and cytoskeletal dynamics as a potential mechanism. These findings enhance our understanding of how mechanical stimulation influences cell behavior and provide new research directions and methodologies for future studies of tendon and ligament mechanobiology.