Research

          Research in the R.R. Fiscus Lab focuses on the identification of novel proteins, the SWITCH in the protein isoforms / SPLICE VARIANTS being expressed, their post-translational modifications (PTMs) and their protein-protein interdependence, which are normally (in healthy individuals) involved in protecting cell survival, promoting regeneration of damaged organs/tissues and preventing the cell damage caused by chronic inflammation, oxidative stress and nitrosative stress (excess iNOS expression that produces toxic-level/toxic-form NO (e.g. Peroxynitrite, ONOO-) & the downstream protein damage via abnormal/pathological S-nitrosylation and tyrosine-nitration.  Oxidative-Nitrosative Stress damages the normal anti-inflammatory/cytoprotective splice variant of PKG-I, i.e. PKG-I-alpha (PKG1alpha). This oxidative-nitrosative damage of PKG1alpha causes loss of its cytoprotective/anti-inflammatory KINASE ACTIVITY, like occurs during the pathogenesis of type-1, type-2 and gestational diabetes/chronic inflammation (from excessively high levels of glucose, fats & cytokines).   Diabetic & chronic inflammation-induced complications can include:    Autism in children of mothers with type-1, type-2 or gestational diabetes, and    (in adults with diabetes and chronic inflammation) :    Alzheimer's disease (AD), erectile dysfunction (ED), liver inflammation, cardiovascular diseases (CVDs), kidney dysfunction and retinal damage (potentially resulting in blindness) and the altered risks of developing certain cancers.   All of these diabetes/chronic inflammation-related pathologies can result, at least in part, from the abnormal damage of essential "cell-survival proteins", such as the key downstream mediator of Insulin Signaling, Akt, and its interdependence ("Reciprocal Activation") with the recently-identified cyto-/neuro-/synapto-protective protein, PKG-I-alpha (see RR Fiscus' Models below & in the Home page, Publications, Patents & Patent Applications, & the background documentation/findings included in Dr. Fiscus' updated Biosketch & CV, provided upon request).  

          Both Akt and PKG-I-alpha are known to be damaged during type-1 and type-2 diabetes (caused by chronic inflammation and the oxidative/nitrosative stress of high glucose and high lipids as well as by excess cytokines ( "Cytokine Storm" ), and the loss of the healthy cytoprotective functions of Akt and PKG-I-alpha in various types of cells (e.g. Brain, Spinal Cord & Peripheral Sensory Neurons, Pancreatic Beta-cells, Vascular Endothelial cells & Stem cells).  In healthy individuals, the protein kinase activities of Akt and PKG-I-alpha continuously play a key role in protecting multiple organ system / cell types against the pathogenesis and progression of many diabetic complications, including AD, ED, liver inflammation, cardiovascular diseases, pancreatic beta-cell dysfunction, and the damage/dysfunctions of the kidney and retina of the eye (see Models below). 

          A special focus of the R.R. Fiscus Lab is identifying novel, previously-unrecognized biological functions of PKG-I-alpha, a splice variant of the well-established vascular protein, protein kinase G (PKG).  Early data from Dr. Fiscus' research serving as Lead Scientist on Protein Kinase Discoveries & Analysis in the lab of Dr. Ferid Murad at Stanford Univ. Med. Sch / Palo Alto VA Med. Ctr. (which contributed to the 1998 NOBEL PRIZE in Physiology & Medicine) during the mid-1980s had established PKG as the key downstream protein kinase  in vascular smooth muscle cells (VSMCs) that is activated by healthy-level NO (both by endogenous endothelium-derived NO, at that time called EDRF, and by therapeutic agents that release or mimic NO, e.g. the NITRATEs) and that in turn mediates the vasodilation and anti-hypertensive effects of NO (Fiscus, Rapaport & Murad, 1983-1984; Fiscus & Murad, 1988; Fiscus, 1988).  Now, it is recognized that PKG can be expressed as three different isoforms (depending on cell type, differentiation/maturation state of the cells and pathological conditions), including PKG-I-alpha and PKG-I-beta (Figure 1), which are splice variants encoded by a common gene, and PKG-II from a separate gene.  

          VSMCs express high levels of both PKG-I-alpha and PKG-I-beta, but not PKG-II.  Interestingly, the sensitive of the two PKG-I splice variants to activation by NO (and its downstream intracellular mediator cGMP) are very different, with PKG-I-alpha being activated by much lower concentrations of NO (e.g. 0.01 - 1 nM, at the lower end of the healthy-level range), whereas PKG-I-beta requires much higher concentrations of NO, estimated to be 10 - 30 times higher than needed for the I-alpha splice variant (Figure 1).  Thus, under healthy physiological conditions, the vascular endothelial cells, which are known to release NO in the 0.01 - 1 nM range under basal conditions and slightly higher NO concentrations when stimulated by eNOS activator ( e.g.   insulin, acetylcholine, bradykinin, estrogen, testosterone, and ApoE2/ApoE3, but not ApoE4 ) would selectively activate the PKG-I-alpha splice variant.  Healthy vascular responses to healthy-level NO that is normally released from eNOS in endothelial cells would involve activation of only the PKG-I-alpha splice variant as the mediator of vasodilations and blood-pressure lowering, i.e. preventing hypertension and many other cardiovascular diseases (see Figure 1).

          The PKG-I-beta splice variant, in contrast, would be activated at much higher concentrations of NO, as would be produced in certain pathologies, such as septic shock and types of severe inflammation, which involves abnormally high levels of NO production from hyperactivated eNOS and the high-level induction of gene expression of the inducible form of NOS ( iNOS ) stimulated by pro-inflammatory cytokines (IL-1-beta, IL-6, TNF-alpha, IFN-gamma).  The model in Figure 1 illustrates the differences in the activation mechanisms for the two splice variants of PKG-I.  The model also shows some of the recognized target proteins for these two PKG-I isoforms.  Because PKG-I-alpha and PKG-I-beta have very different subcellular distributions, which place these two isoforms in contact with very different subsets of downstream target proteins, the actual proteins that get phosphorylated by PKG-I-alpha and PKG-I-beta are very different, thus mediating very different biological effects, e.g. increased survival and increased proliferation of cells in the case of PKG-I-alpha activation, versus decreased cell survival (induction of  apoptosis) and decreased proliferation (inhibition of cell cycle progression) of cells in the case of PKG-I-beta activation (Figure 1). 

Figure 1.  Model showing the opposite biological effects of the two splice variants of PKG-I on cell survival and proliferation.  PKG-I-alpha and PKG-I-beta typically have very different subcellular localizations, placing these two protein kinases in contact with a different subset of downstream target proteins, thus resulting in the phosphorylation of different proteins that have different biological effects (in this case, opposite effects).  This can explain the apparent contradictory data presented in the literature concerning the role of PKG in mediating changes in cell survival and proliferation.  Early studies did not distinguish between the different isoforms of PKG and had used relatively high levels of stimulation [higher levels of NO donors or high concentrations of phosphodiesterase type-5 (PDE-5) inhibitors like Exisulind, which would  cause very large increases in intracellular levels of cGMP and would activate not only PKG-I-alpha but also PKG-I-beta].  In this situation, the growth-inhibition and induction of apoptosis caused by high-level activation of PKG-I-beta would predominate over the growth-promoting and pro-cell-survival/anti-apoptosis  effects mediated by PKG-I-alpha.  More recent studies, primarily by the R.R. Fiscus Lab, have shown that lower-level stimulation by "healthy-level NO", which would selectively activate only the PKG-I-alpha splice variant, results in increased cell survival and proliferation, a condition that is typically happening in cell culture experiments where serum factors are continuously stimulating the endogenous eNOS and/or nNOS activity with downstream activation of PKG-I-alpha (see Model in Figure 1).  This PKG-I-alpha activation in cultured cells exposed to serum factors (or other growth-promoting factors, e.g. insulin) plays an important role in the stimulation of cell proliferation and cell survival of these cells, as established in experiments using siRNA gene knockdown and kinase inhibitors (e.g. shown in vascular endothelial and smooth muscle cells, pancreatic beta-cells, bone marrow-derived mesenchymal (stromal) stem cells and various types of cancer cells) in experiments conducted in the R.R. Fiscus Lab (see Publications and discussion below).

Not shown in the model are additional data from the R.R. Fiscus Lab establishing a neuroprotective/neuro-regenerative role of PKG in brain neurons and other neural cells, first illustrated in our collaborative research with the Mark Mattson lab (with experiments conducted by Steven Barger) in Sanders-Brown Center on Aging, University of Kentucky (Barger, Fiscus, Ruth, Hofmann & Mattson, J. Neurochem. 1995).  This early study showed a key role of PKG in brain hippocampal neurons that is necessary for protecting these neurons from the neurotoxicity of excess glutamate (model of Alzheimer's disease).  Many subsequent studies from the R.R. Fiscus Lab have now shown that it is the PKG-I-alpha splice variant in neural cells that mediates the neuroprotective and neuro-regenerative effects of healthy-level NO, as would be provided by healthy cerebrovascular endothelial cells in response to eNOS activation by physiological levels of insulin, acetylcholine and glutamate (see Models below and our publications, e.g.  Fiscus, 2002;  Fiscus et al., 2002;  Johlfs and Fiscus, 2010;  Fiscus and Johlfs, 2012 - book chapter in Protein Kinase Technologies-Neuromethods "Protein kinase G (PKG): Involvement in promoting neural cell survival, proliferation, synaptogenesis and synaptic plasticity and the use of new ultrasensitive capillary-electrophoresis-based methodology for measuring PKG expression and molecular actions").  This new capillary-electrophoresis-based methodology, utilizing capillary isoelectric focusing (cIEF technology, i.e. NanoPro 1000 system, discussed in Expertise section on our home page) has allowed identification of PKG-I-alpha as the predominate splice variant of PKG in most neural cells (see data and discussion in Fiscus and Johlfs, 2012), illustrating a key role of PKG-I-alpha in promoting CREB activation that enhances learning and memory and VASP phosphorylation that protects against excessive inflammation, all helping to prevent the pathogenesis of Alzheimer's disease.

The model also shows the effects of c-Src activation (stimulated by insulin, EGF, fetal serum factors, and other growth/survival factors) that causes tyrosine-phosphorylation of PKG-I-alpha, which in turn enhances PKG-I-alpha's basal activity and sensitivity to cGMP-induced activation (publications from R.R. Fiscus Lab:  Leung et al., 2010;  Fiscus & Johlfs, book chapter in Protein Kinase Technologies-Neuromethods, 2012;  Fiscus et al., book chapter in Ovarian Cancer - Basic Science Perspectives, 2012).  In cancer cells, which often have over-expressed c-Src and/or hyperactivated c-Src, the tyrosine-phosphorylation of PKG-I-alpha would be exaggerated, resulting in high/pathological-level activation (i.e. hyperactivation) of PKG-I-alpha, resulting in exaggerated cell proliferation and cell survival [i.e. resistance to chemotherapy (chemoresistance)], as shown in our publications with brain, lung and ovarian cancers (Johlfs and Fiscus, 2010;  Leung et al., 2010;  Wong and Fiscus, 2012).   We have also shown that c-Src is also a downstream target protein of PKG-I-alpha, being phosphorylated on serine-17 by PKG-I-alpha, which enhances c-Src's tyrosine kinase activity and likely alters c-Src's translocation and phosphorylation capabilities within cells (Fiscus and Johlfs, 2011).  The R.R. Fiscus Lab has proposed that this exaggerated "reciprocal phosphorylation/activation" between c-Src and PKG-I-alpha explains the hyperactivation of PKG-I-alpha observed in many types of cancer cells (in fact, all of the 25 different cancer cell lines, including brain, breast, lung, ovarian, pancreatic and prostate cancers, studied in the R.R. Fiscus Lab over the last 10 years).

           The R.R. Fiscus Lab has shown that the PKG1alpha (PKG-I-alpha) splice variant is essential for mediating pro-cell-survival  and proliferation in various neural cells (e.g. NG108-15 cells, N1E-115 neuroblastoma cells), pancreatic beta-cells (including INS-1, beta-TC-6, RINm5F and the recently developed human, insulin-secreting beta-cell line 1.1B4) and bone-marrow-derived stromal/mesenchymal stem cells (BM-MSCs, using OP9 cells as a model).  (see our published research articles & Book chapters in Publications section)