Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization

Critical to homeostasis of blood cell production by hematopoietic stem/progenitor (HSC/P) cells is the regulation of HSC/P retention within the bone marrow microenvironment and migration between the bone marrow and the blood. Key extracellular regulatory elements for this process have been defined (cell–cell adhesion, growth factors, chemokines), but the mechanism by which HSC/P cells reconcile multiple external signals has not been elucidated. Rac and related small GTPases are candidates for this role and were studied in HSC/P deficient in Rac2, a hematopoietic cell-specific family member. Rac2 appears to be critical for HSC/P adhesion both in vitro and in vivo, whereas a compensatory increase in Cdc42 activation regulates HSC/P migration. This genetic analysis provides physiological evidence of cross-talk between GTPase proteins and suggests that a balance of these two GTPases controls HSC/P adhesion and mobilization in vivo.

Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor

Whereas several apoptosis-related proteins have been linked to the antiapoptotic effects of Akt serine–threonine kinase, the search continues to explain the Akt signaling role in promoting cell survival via antiapoptotic effects. Here, we demonstrate that Akt phosphorylates the androgen receptor (AR) at Ser-210 and Ser-790. A mutation at AR Ser-210 results in the reversal of Akt-mediated suppression of AR transactivation. Activation of the phosphatidylinositol-3-OH kinase/Akt pathway results in the suppression of AR target genes, such as p21, and the decrease of androgen/AR-mediated apoptosis, which may involve the inhibition of interaction between AR and AR coregulators. Together, these findings provide a molecular basis for cross-talk between two signaling pathways at the level of Akt and AR–AR coregulators that may help us to better understand the roles of Akt in the androgen/AR-mediated apoptosis.

A Functional Calvin Cycle Is Not Indispensable for the Light Activation of C4 Phosphoenolpyruvate Carboxylase Kinase and Its Target Enzyme in the Maize Mutant bundle sheath defective2-mutable11

We used a pale-green maize (Zea mays L.) mutant that fails to accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to test the working hypothesis that the regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) by its Ca2+-insensitive protein-serine/threonine kinase (PEPC kinase) in the C4 mesophyll cytosol depends on cross-talk with a functional Calvin cycle in the bundle sheath. Wild-type (W22) and bundle sheath defective2-mutable1 (bsd2-m1) seeds were grown in a controlled environment chamber at 100 to 130 μmol m−2 s−1 photosynthetic photon flux density, and leaf tissue was harvested 11 d after sowing, following exposure to various light intensities. Immunoblot analysis showed no major difference in the amount of polypeptide present for several mesophyll- and bundle-sheath-specific photosynthetic enzymes apart from Rubisco, which was either completely absent or very much reduced in the mutant. Similarly, leaf net CO2-exchange analysis and in vitro radiometric Rubisco assays showed that no appreciable carbon fixation was occurring in the mutant. In contrast, the sensitivity of PEPC to malate inhibition in bsd2-m1 leaves decreased significantly with an increase in light intensity, and there was a concomitant increase in PEPC kinase activity, similar to that seen in wild-type leaf tissue. Thus, although bsd2-m1 mutant plants lack an operative Calvin cycle, light activation of PEPC kinase and its target enzyme are not grossly perturbed.

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca2+]cyt response. Cyclic-nucleotide-mediated elevations in [Ca2+]cyt involved both internal and external Ca2+ stores. Both cAMP- and cGMP-mediated [Ca2+]cyt elevations could be inhibited by the Ca2+-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca2+]cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca2+]cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca2+ signaling.

Communication between noncontacting macromolecules

We present a quantitative experimental demonstration of solvent-mediated communication between noncontacting biopolymers. We show that changes in the activity of a solvent component brought about by a conformational change in one biopolymer can result in changes in the physical properties of a second noncontacting biopolymer present in solution. Specifically, we show that the release of protons on denaturation of a donor polymer (in this case, a four-stranded DNA tetraplex, iDNA) modulates the melting temperature of a noncontacting, acceptor polymer [in this case poly(A)]. In addition to such proton-mediated cross talk, we also demonstrate counterion-mediated cross talk between noncontacting biopolymers. Specifically, we show that counterion association/release on denaturation of native salmon sperm DNA (the donor polymer) can modulate the melting temperature of poly(dA)⋅poly(dT) (the acceptor polymer). Taken together, these two examples demonstrate how poly(A) and poly(dA)⋅poly(dT) can serve as molecular probes that report the pH and free salt concentrations in solution, respectively. Further, we demonstrate how such through-solvent dialogue between biopolymers that do not directly interact can be used to evaluate (in a model-free manner) association/dissociation reactions of solvent components (e.g., protons, sodium cations) with one of the two biopolymers. We propose that such through-solution dialogue is a general property of all biopolymers. As a result, such solvent-mediated cross talk should be considered when assessing reactions of multicomponent systems such as those that exist in essentially all biological processes.

An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells.

Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.

Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor.

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.

Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53.

Thyroid hormone nuclear receptors (TRs) are ligand-dependent transcriptional factors that regulate growth, differentiation, and development. The molecular mechanisms by which TRs mediate these effects are unclear. One prevailing hypothesis suggests that TRs may cooperate with other transcriptional factors to mediate their biological effects. In this study, we tested this hypothesis by examining whether the activity of TRs is modulated by the tumor suppressor p53. p53 is a nuclear protein that regulates gene expression via sequence-specific DNA binding and/or direct protein-protein interaction. We found that the human TR subtype beta 1 (h-TR beta 1) physically interacted with p53 via its DNA binding domain. As a result of this physical interaction, binding of h-TR beta 1 to its hormone response elements either as homodimer or as a heterodimer with the retinoic X receptor was inhibited by p53 in a concentration-dependent manner. In transfected cells, wild-type p53 repressed the hormone-dependent transcriptional activation of h-TR beta 1. In contrast, mutant p53 either had no effect or activated the transcriptional activity of h-TR beta 1 depending on the type of hormone response elements. These results indicate the gene regulating activity of TRs was modulated by p53, suggesting that the cross talk between these two transcriptional factors may play an important role in the biology of normal and cancer cells.

Basolateral membrane Na+/H+ exchange enhances HCO3- absorption in rat medullary thick ascending limb: evidence for functional coupling between basolateral and apical membrane Na+/H+ exchangers.

The role of basolateral membrane Na+/H+ exchange in transepithelial HCO3- absorption (JHCO3) was examined in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. In Na(+)-free solutions, addition of Na+ to the bath resulted in a rapid, amiloride-sensitive increase in intracellular pH. In MTALs perfused and bathed with solutions containing 146 mM Na+ and 25 mM HCO3-, bath addition of amiloride (1 mM) or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 microM) reversibly inhibited JHCO3 by 50%. Evidence that the inhibition of JHCO3 by bath amiloride was the result of inhibition of Na+/H+ exchange included the following: (i) the IC50 for amiloride was 5-10 microM, (ii) EIPA was a 50-fold more potent inhibitor than amiloride, (iii) the inhibition by bath amiloride was Na+ dependent, and (iv) significant inhibition was observed with EIPA as low as 0.1 microM. Fifty micromolar amiloride or 1 microM EIPA inhibited JHCO3 by 35% when added to the bath but had no effect when added to the tubule lumen, indicating that addition of amiloride to the bath did not directly inhibit apical membrane Na+/H+ exchange. In experiments in which apical Na+/H+ exchange was assessed from the initial rate of cell acidification following luminal EIPA addition, bath EIPA secondarily inhibited apical Na+/H+ exchange activity by 46%. These results demonstrate basolateral membrane Na+/H+ exchange enhances transepithelial HCO3- absorption in the MTAL. This effect appears to be the result of cross-talk in which an increase in basolateral membrane Na+/H+ exchange activity secondarily increases apical membrane Na+/H+ exchange activity.

Investigating the Effect of Histone H4 Sumoylation on Chromatin Structure and Function

Thesis (Ph.D.)--University of Washington, 2016-06

Systematic proteomic strategies to map the human signaling landscape

Thesis (Ph.D.)--University of Washington, 2016-05

Additional signalling compounds are required to orchestrate plant development

Plants are necessarily complex systems that require monitoring of multiple environmental signals and, in response to those signals, coordination of differentiation and development of an extensive array of cell types at multiple locations. This coordination must rely on integration of long-distance signals that provide a means of communication among different plant parts. We propose that the relatively well-characterized classical phytohormones must act with several other long-distance signals to achieve this level of organization with dynamic yet measured responses. This is supported by observations that classical phytohormones: (i) operate in complex yet experimentally unresolved networks involving cross-talk and feedback, (ii) are generally multifunctional and nonspecific and hence must rely on other long-distance cues or pre-set conditions to achieve specificity and (iii) are likely to mask roles of other long-distance signals in several experimental contexts. We present evidence for involvement of novel long-distance signals in three developmental processes-branching, flowering and nodulation, and discuss the possible identities of novel signalling molecules.

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

Arachidonic acid release from mammalian cells transfected with human groups IIA and X secreted phospholipase A(2) occurs predominantly during the secretory process and with the involvement of cytosolic phospholipase A(2)-alpha

Stable expression of human groups IIA and X secreted phospholipases A(2) (hGIIA and hGX) in CHO-K1 and HEK293 cells leads to serum- and interleukin-1beta-promoted arachidonate release. Using mutant CHO-K1 cell lines, it is shown that this arachidonate release does not require heparan sulfate proteoglycan- or glycosylphosphatidylinositol-anchored proteins. It is shown that the potent secreted phospholipase A(2) inhibitor Me-Indoxam is cell-impermeable. By use of Me-Indoxam and the cell-impermeable, secreted phospholipase A(2) trapping agent heparin, it is shown that hGIIA liberates free arachidonate prior to secretion from the cell. With hGX-transfected CHO-K1 cells, arachidonate release occurs before and after enzyme secretion, whereas all of the arachidonate release from HEK293 cells occurs prior to enzyme secretion. Immunocytochemical studies by confocal laser and electron microscopies show localization of hGIIA to the cell surface and Golgi compartment. Additional results show that the interleukin-1beta-dependent release of arachidonate is promoted by secreted phospholipase A(2) expression and is completely dependent on cytosolic (group IVA) phospholipase A(2). These results along with additional data resolve the paradox that efficient arachidonic acid release occurs with hGIIA-transfected cells, and yet exogenously added hGIIA is poorly able to liberate arachidonic acid from mammalian cells.

The challenges of abundance: epithelial junctions and small GTPase signalling

Small GTPases of the Ras superfamily play critical roles in epithelial biogenesis. Many key morphogenetic functions occur when small GTPases act at epithelial junctions, where they mediate an increasingly complex interplay between cell-cell adhesion molecules and fundamental cellular processes, such as cytoskeletal activity, polarity and trafficking. Important recent advances in this field include the role of additional members of the Ras superfamily in cell-cell contact stability and the capacity for polarity determinants to regulate small GTPase signalling. Interestingly, small GTPases may participate in the cross-talk between different adhesive receptors: in tissues classical cadherins can selectively regulate other junctions through cell signalling rather than through a global influence on cell-cell cohesion.