Calcium ([Ca2+]i) oscillations, a hallmark of mammalian fertilization, are essential to induce egg activation and embryonic development. As the zygotes transition through the cell cycle, these [Ca 2+]i oscillations progressively diminish until they cease in interphase zygotes. While the mechanism(s) underlying the regulation of [Ca2+]i oscillations may be multi-layered, inositol 1,4,5-trisphosphate receptors (IP3Rs) emerge as a focal point in this regulation. IP3Rs, the calcium channels expressed in all cell types and abundantly expressed in mammalian eggs, contain consensus sequences for phosphorylation by various kinases and interact with members of the cytoskeleton, serving as an integrator of regulatory signals. This dissertation highlights the impact of biochemical and cellular changes in IP3R-1 on fertilization-induced [Ca2+]i oscillations. Together with the changes in the cellular distribution of the sperm factor, these cell cycle-dependent modifications in IP3R-1 may underlie the regulation of [Ca 2+]i oscillations in fertilized mammalian eggs.

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Rationale: The pathogenesis and progression of pressure-overload heart failure (HF) encompasses aberrations in gene regulation, leading to maladaptive cardiac hypertrophy, ventricular remodeling, and contractile dysfunction. The trigger for maladaptation and HF is signaling through the G protein, Gq, and one downstream effector for this pathway is activation of non-canonical activity of G protein-coupled receptor kinase-5 (GRK5). This kinase, following hypertrophic stimuli, can translocate and accumulate in the nucleus of cardiomyocytes. The nuclear targeting of GRK5 is mediated by an amino-terminal (NT) domain that can bind calmodulin (CaM), which is required before its nuclear translocation. Objective: This study attempted to thwart GRK5-mediated pathology in pressure-overload maladaptation and HF by cardiomyocyte expression of a peptide encoding the NT of GRK5 (GRK5nt) that includes this CaM binding domain. Methods and Results: In vitro studies in myocytes showed that Gq-coupled receptor mediated hypertrophy was abrogated with GRK5nt expression and this included attenuation of pathological gene expression and NFAT activity. We confirmed that the GRK5nt binds to Ca2+-CaM, prevents its association with endogenous GRK5, and prevents its nuclear translocation. We generated cardiac-specific GRK5nt transgenic mice and showed in vivo that expression of this peptide prevents hypertrophic nuclear translocation of GRK5 and these mice exhibit significantly less cardiac hypertrophy, ventricular dysfunction, pulmonary congestion, and cardiac fibrosis following chronic transverse aortic constriction. Conclusions: Together our data support a role for GRK5nt as an inhibitor of pathological nuclear GRK5 signaling for HF prevention. / Biomedical Sciences

In this study, we assessed the contributions of plasma membrane (PM) microdomain targeting to the functions of H-Ras and R-Ras. These paralogues have identical effector-binding regions, but variant C-terminal targeting domains (tDs) which are responsible for lateral microdomain distribution: activated H-Ras targets to lipid ordered/disordered (Lo/Ld) domain borders, and R-Ras to Lo domains (rafts). We hypothesized that PM distribution regulates Ras effector interactions and downstream signaling. We used tD swap mutants, and assessed effects on signal transduction, cell proliferation, transformation, and tumorigenesis. R-Ras harboring the H-Ras tD (R-Ras-tH) interacted with Raf, and induced Raf and ERK phosphorylation similar to H-Ras. R-Ras-tH stimulated proliferation and transformation in vitro, and these effects were blocked by both MEK and PI3K inhibition. Conversely, the R-Ras tD suppressed H-Ras-mediated Raf activation and ERK phosphorylation, proliferation, and transformation. Thus, Ras access to Raf at the PM is sufficient for MAPK activation and is a principal component of Ras mitogenesis and transformation. Fusion of the R-Ras extended N-terminal domain to H-Ras had no effect on proliferation, but inhibited transformation and tumor progression, indicating that the R-Ras N-terminus also contributes negative regulation to these Ras functions. PI3K activation was tD-independent; however, H-Ras was a stronger activator of PI3K than R-Ras, with either tD. PI3K inhibition nearly ablated transformation by R-Ras-tH, H-Ras, and H-Ras-tR, whereas MEK inhibition had a modest effect on Ras-tH-driven transformation but no effect on H-Ras-tR transformation. R-Ras-tH supported tumor initiation, but not tumor progression. Whereas H-Ras-tR-induced transformation was reduced relative to H-Ras, tumor progression was robust and similar to H-Ras. H-Ras tumor growth was moderately suppressed by MEK inhibition, which had no effect on H-Ras-tR tumor growth. In contrast, PI3K inhibition markedly suppressed tumor growth by H-Ras and H-Ras-tR, indicating that sustained PI3K signaling is a critical pathway for H-Ras-driven tumor progression, independent of microdomains. In the second phase of the study, we investigated the combinatorial use of two drugs currently either in active use as anti-cancer agents (Rapamycin) or in clinical trials (OTX008), as a novel strategy to inhibit H-Ras-driven tumor progression. H-Ras anchored to the plasma membrane shuttles from the lipid ordered (Lo) domain to the lipid ordered/lipid disordered border upon activation, and retention of H-Ras at these sites requires Galectin-1 (Gal-1). We have previously found that genetically-mediated Lo sequestration of H-Ras inhibited MAPK signaling but not PI3K activation. Here we show that inhibition of Gal-1 with OTX008 sequestered H-Ras in the Lo domain, blocked H-Ras-mediated MAPK signaling, and attenuated H-Ras-driven tumor progression in mice. H-Ras-driven tumor growth was also attenuated by treatment with mTOR inhibitor Rapamycin, and this effect was further enhanced in tumors driven by Lo-sequestered H-Ras. These drugs also revealed bidirectional cross-talk in H-Ras pathways. Moreover, dual pathway inhibition with OTX008 and Rapamycin resulted in nearly complete ablation of H-Ras-driven tumor growth. These findings indicate that membrane microdomain sequestration of H-Ras with OTX008, coupled with mTOR inhibition, may support a novel therapeutic approach to treat H-Ras mutant cancers. / Cell Biology

Osteoactivin (OA) is a novel osteogenic and repair factor. It has the ability to regulate cell proliferation, adhesion, differentiation, and synthesis and regulation of extracellular matrix proteins in various cell types under both normal and pathological conditions. Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased 3-fold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation ex vivo. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts ex vivo was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-β1 and TGF-β receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. OA expression increases during tissue degeneration and regeneration, fracture repair, and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA’s expression with repetitive overuse injuries is unknown. We sought to evaluate in an animal model of upper extremity repetitive overuse, at low force loads: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72), a protein known to increase during metabolic stress and be involved in cellular repair. We hypothesized that OA is functioning as a growth factor during periods of tissue repair. Young adult, female Sprague-Dawley rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks, and were compared to control rats. Quantitative PCR, Western blot analyses and immunohistochemistry showed increased OA mRNA and protein expression in flexor digitorum muscles of 6-week HRNF rats, compared to controls. OA protein levels increased similarly in 6-week HRNF flexor digitorum tendons. Increased OA immunostaining was localized to the myofiber sarcolemma, macrophage-like cells and tenocytes. In muscles, Western blot analyses showed progressive increases in MMP-1, -2 and -3, whereas tendons had increased MMP-1 and -3, with HRNF task performance. ELISA and immunohistochemistry showed increased HSP72 in 6-week HRNF muscles, and co-localization with OA in the myofiber sarcolemma. HSP72 increased in 6-week HRNF tenocytes, compared to controls. Inflammatory cytokines IL-1alpha or beta showed transient increases at 3 weeks in muscles and tendons, while IL-1alpha was significantly decreased in 6-week HRNF muscles. The simultaneous increases of MMPs and HSP72 with OA, factors involved in tissue repair, supports a role of OA in tissue regeneration after repetitive overuse. We extended the study above to examine the expression of OA during high repetition high force loading in our animal model of upper extremity overuse, in combination with anti-inflammatory drug, to evaluate OA’s link to inflammatory processes. Young adult female rats underwent an initial training period to learn the task (10 min/day, 5 days/wk, for 6 wks), before then performing a high repetition high force (HRHF) task for 11 weeks (2 hours/day, 3 days/week). Results were compared to age-matched control (C) rats. At the end of HRHF task week 3, two cohorts of HRHF rats received 5 intraperitoneal injections of saline (HRHF+Veh) or anti-rat TNF-a (HRHF+anti-TNF) across 4-7 weeks, as did controls (C+Veh and C+anti-TNF). Two other cohorts rested during weeks 4-7 with or without treatment (HRHF+anti-TNF/Rest and HRHF+Veh/Rest), to parallel its partner group. Motor behavior was assessed and revealed decreased grip strength in HRHF+Veh rats beginning immediately post training (HRHF task week 0), and that anti-TNF-α treatment prevented this grip strength decline. The 4-week anti-TNF-α therapy extended maintenance of grip strength near control levels through week 9, despite no further treatment after week 7. By experimental week 11, ELISA showed no significant differences in OA levels in forearm flexor digitorum muscles, and histomorphometry showed no difference in the circumference of this muscle in any HRHF group, compared to controls, matching findings of no gain in grip strength above control levels in any HRHF group. However, ELISA of distal radius and ulna homogenates showed increased OA levels in HRHF+anti-TNF rats, as well as increased IL-18 in bones of both anti-TNF treated HRHF groups (HRHF+anti-TNF and HRHF+anti-TNF/Rest rats), compared to controls. Micro-CT analysis showed that rats receiving anti-TNF-α treatment, with or without rest, had increased bone mass (detected as increased trabecular bone volume, thickness, and number and reduced trabecular separation), compared to the other groups. Histomorphometry showed increased osteoblast numbers in HRHF+anti-TNF rats, compared controls, yet decreased osteoclast numbers, compared to HRHF+Veh rats, indicative of increased bone anabolism in anti-TNF-a treated rats. Thus, these findings suggest that TNF-α blocks OA expression in bones, and that its increase when combined with prolonged repetitive loading, enhances osteoblast activity and bone formation. / Cell Biology

As the principal cellular agents initiating thrombus (clot) formation, the primary function of platelets is maintenance of blood hemostasis. These anucleate cell fragments lack nuclear DNA and so provide a unique model for investigating post-transcriptional regulation of gene expression via RNA interference (RNAi). Although platelets are not capable of transcribing nascent mRNA, they are able to maintain protein homeostasis throughout their ~10-day lifespan in circulation; how platelets are able to do so, considering they contain a functional proteasome, remain unclear. We hypothesized that platelet protein translation and homeostasis is driven by ongoing plasma-derived growth factor signaling balanced with degradation, which is regulated by Argonaute 2 (Ago2)-mediated RNAi. In nucleated cells, RNAi occurs via RNA-induced silencing complexes (RISC), in which a guide RNA such as a microRNA (miRNA) docked in an effector protein, typically Ago2, recruits a target mRNA via guide:target sequence complementarity and results in suppression of mRNA translation via multiple mechanisms. Though RNAi has not yet been fully explored in the context of anucleate cells, platelets contain the machinery and proteins necessary for RNAi as well as being highly enriched in a diverse array of miRNAs. Additionally, variations in platelet miRNAs in humans are associated with variable platelet reactivity and risk of thrombosis.   We have found platelets to be capable of taking up exogenous double-stranded miRNA and have published on their subsequent ability to utilize the miRNA to modulate signal-activated translation and platelet function (Aim 1). In order to investigate the role of constitutive translation in circulating platelets (Aim 2) and the effect of Ago2 in regulation of platelet reactivity (Aim 3) we generated a novel platelet-specific Ago2 deleted (Ago2fl/fl/Pf4-Cre) mouse line and compared the proteome of knockout (KO) mice to wild-type (WT), by sex. Based on Ago2 roles in RNAi and miRNA enrichment in platelets, we predicted large-scale increases in platelet protein expression with megakaryocytic Ago2 deletion. However, only a small subset of genes was significantly upregulated. Among these was Ago1, which was significantly increased in both male and female Ago2 KO platelets. These results strongly suggest that Ago-mediated RNAi is essential for platelet development. Interestingly, deletion of platelet Ago2 resulted in decreased protein expression of a different subset of genes in platelet from female mice only; this sex-specific effect may be due to hormonal differences, namely estrogen, as many of these regulated proteins are encoded by estrogen-responsive genes. The gene set downregulated in Ago2 KO female platelets vs WT, but not males, corresponded to genes known to be coordinately regulated at the transcriptional level by ERβ in conjunction with Ago2 in breast cancer cells. These results point to a novel role for Ago2 in modulating the platelet transcriptome in megakaryocytes, affecting the mRNAome in the platelets they generate, in a hormone-dependent manner.   Platelets from both male and female KO mice had significantly increased mean platelet volumes (MPV), not explained by changes in platelet production, lifespan, or protein content; however, KO megakaryocytes (MKs) had significantly increased size & ploidy, accounting for increased MPV, although femurs in the KO mice had fewer MKs. Furthermore, KO mice exhibited faster clot retraction and increased surface expression of integrin αIIbβ3. Platelets from male KO mice showed increased αIIbβ3 integrin activity and P-selectin expression following thrombin and U46619 stimulation, a thromboxane analogue, but these effects were not seen with collagen or ADP, pointing to agonist pathway-specific effects. Bioinformatics analysis revealed sex-specific changes in the platelet proteome of KOs to account for this change in reactivity; a cluster of mitochondrial enzymes modulated in male KOs only, involved in ROS metabolism and respiration, may be responsible for the observed difference in the thromboxane response. Taken together, our findings suggest Ago2-mediated RNAi to be an important regulator of megakaryopoeisis and platelet reactivity. / Biomedical Sciences