Biology

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Stanniocalcin-1 is a naturally occurring L-channel inhibitor in cardiomyocytes: relevance to human heart failure
(2003-07-01) Sheikh-Hamad, David; Bick, Roger; Gang-Yi, Wu; Monster Christensen, Birgitte; Razeghi, Peter; Poindexter, Brian; Taegtmeyer, Heinrich; Wamsley, Ann; Padda, Ranjit; Entman, Mark; Nielsen, Soren; Youker, Keith
Cardiomyocytes of the failing heart undergo profound phenotypic and structural changes that are accompanied by variations in the genetic program and profile of calcium homeostatic proteins. The underlying mechanisms for these changes remain unclear. Because the mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) is expressed in the heart, we reasoned that STC1 might play a role in the adaptive-maladaptive processes that lead to the heart failure phenotype. We examined the expression and localization of STC1 in cardiac tissue of patients with advanced heart failure before and after mechanical unloading using a left ventricular assist device (LVAD), and we compared the results with those of normal heart tissue. STC1 protein is markedly upregulated in cardiomyocytes and arterial walls of failing hearts pre-LVAD and is strikingly reduced after LVAD treatment. STC1 is diffusely expressed in cardiomyocytes, although nuclear predominance is apparent. Addition of recombinant STC1 to the medium of cultured rat cardiomyocytes slows their endogenous beating rate and diminishes the rise in intracellular calcium with each contraction. Furthermore, using whole cell patch-clamp studies in cultured rat cardiomyocytes, we find that addition of STC1 to the bath causes reversible inhibition of transmembrane calcium currents through L-channels. Our data suggest differential regulation of myocardial STC1 protein expression in heart failure. In addition, STC1 may regulate calcium currents in cardiomyocytes and may contribute to the alterations in calcium homeostasis of the failing heart. stanniocalcin-1 (STC1) is a homodimeric glycoprotein hormone involved in calcium regulation in bony fish (8), where elevation of serum calcium triggers the release of STC1 from the corpuscles of Stannius (23), organs associated with the kidneys (26). On circulation in the gill and intestine, STC1 inhibits calcium flux from the aquatic environment through these organs, thus maintaining normal calcium concentrations in the blood (15, 24). In mammals, STC1 is expressed in multiple organs, including the heart, skeletal muscle, brain, thyroid, spleen, thymus, parathyroid, lung, kidney, pancreas, small intestine, colon, placenta, ovary, testes, and prostate (4, 5, 22). The wide expression of STC1 suggested that it might function in an autocrine and/or paracrine manner, whereas its localization to the heart and skeletal muscle suggested a role in myocyte function. Through the evolutionary process from fish to mammals, STC1 appears to have maintained its functional role in calcium regulation, because mammalian STC1 appears to be involved in calcium homeostasis in the normal physiology of the gut (16) and in the adaptive response of brain cells to ischemic injury (28). Because cardiomyocyte calcium homeostasis demonstrates a wide range of abnormalities in patients with heart failure (2, 9, 11, 13, 17, 21), we hypothesized that myocardial expression of STC1 may be relevant to calcium homeostasis in the failing heart. Our current data suggest differential expression of STC1 protein in cardiomyocytes and blood vessel walls of failing hearts and are consistent with a potential role for STC1 in cardiomyocyte calcium homeostasis.
AT1A-mediated activation of kidney JNK1 and SMAD2 in obstructive uropathy: preservation of kidney tissue mass using candesartan
(2004-09-01) Wamsley-Davis, Ann; Padda, Ranjit; Truong, Luan D.; Tsao, Chun Chui; Zhang, Ping; Sheikh-Hamad, David
Literature suggests the involvement of the renin-angiotensin system and transforming growth factor (TGF)-β in the renal injury that follows chronic ureteric obstruction. SMAD proteins and the JNK1 cascade are essential components of TGF-β signaling machinery, and recent data suggest cooperative interaction between JNK1 and SMAD proteins in TGF-β-mediated gene expression. We used a rat model of chronic unilateral ureteric obstruction to study the effects of candesartan, an AT1A-receptor blocker, on tissue morphology and the activities of JNK1 and SMAD2 protein in the kidney. Ureteric obstruction for 28 days leads to interstitial fibrosis, tubule atrophy, and marked activation of SMAD2 and JNK1, without significant change in p38 kinase or ERK. Candesartan treatment, however, attenuated the chronic tubulointerstitial injury in obstructed kidneys and was associated with significant preservation of kidney tissue mass. Furthermore, treatment with candesartan diminished JNK1 activity and downregulated SMAD2 protein and activity in obstructed kidneys. In conclusion, obstructed kidneys showed chronic tubulointerstitial injury, which was associated with JNK1 and SMAD2 activation. The renoprotective effects afforded by AT1A-receptor blockade in obstructive uropathy are consistent with attenuation of JNK1- and SMAD2-mediated renal injury. obstruction of urine flow complicates many human diseases, such as prostate hypertrophy, abdominopelvic malignancies, and retroperitoneal fibrosis. It is characterized by infiltration of mononuclear inflammatory cells, predominantly macrophages, and is accompanied by activation of cytokines, growth factors, and mediators of apoptosis, the net result of which is the deletion of tubular cells by apoptosis and replacement of renal parenchyma with fibrous tissue. If left untreated, chronic kidney obstruction leads to loss of functional renal parenchyma, and ultimately, the development of kidney failure. Numerous reports implicate the renin-angiotensin system in the pathogenesis of ureteric obstruction, and inhibition of the rennin-angiotensin system, using either angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers, has been shown to attenuate the renal injury that follows ureteric obstruction (19, 23, 37). Activation of the kidney renin-angiotensin system occurs immediately after urteric obstruction, with resultant vasoconstriction and salt and water retention (14). These hemodynamic changes are followed by the activation of a number of cytokines (6, 9, 20, 21, 36, 50), some of which have been directly linked to angiotensin II, such as monocyte chemotactic protein-1 (MCP-1) (20) and transforming growth factor-β (TGF-β) (22), which appears to play a key role in the genesis of the ensuing fibrosis (32). TGF-β signaling is initiated after ligand binding to the TGF-β receptor (15, 33), leading to phosphorylation of SMAD2 or SMAD3 (1, 47, 53). SMAD proteins represent a group of transcription factors involved in gene regulation downstream of the TGF-β receptor. A heteromeric SMAD proteins complex is formed (7), resulting in translocation of the complex to the nucleus (1, 31, 47), where it can interact with transcription factors directly (7), or indirectly (27), to regulate gene expression. It was recently reported that JNK pathway (25, 51), which targets the activation of c-Jun and activated transcription factor-2 (ATF-2) (8, 12, 16, 26), is stimulated rapidly by TGF-β in human fibrosarcoma cells and that JNK activity is essential for TGF-β-induced fibronectin production (17). Furthermore, it was also suggested that SMAD proteins and the JNK1 pathway interact cooperatively in TGF-β signaling (17). These observations assume particular importance, in light of recent data that implicate JNK1 in the development of cell apoptosis after ischemia and UV irradiation in a variety of tissues (2, 3). Apoptosis is a crucial component in the pathogenesis of ureteric obstruction (5, 49). Based on these observations, we hypothesized that JNK1 may be a key contributor to the development of renal injury and fibrosis in obstructive uropathy, and thus renal protection after AT1A-receptor blockade in obstructive uropathy may be related to the downregulation of JNK1 and SMAD proteins. Consistent with this hypothesis, our data suggest activation of SMAD2 and JNK1 signaling in obstructed kidneys in a manner that is AT1A dependent. AT1A- receptor blockade using candesartan downregulates JNK1 and SMAD2, decreases tissue fibrosis, and leads to tissue preservation in obstructed kidneys.
MEKK3-mediated signaling to p38 kinase and TonE in hypertonically stressed kidney cells
(2006-10-01) Padda, Ranjit; Wamsley-Davis, Ann; Gustin, Michael C.; Ross, Rebekah; Yu, Christina; Sheik-Hamad, David
Mitogen-activated protein kinase (MAPK) cascades contain a trio of kinases, MAPK kinase kinase (MKKK) → MAPK kinase (MKK) → MAPK, that mediate a variety of cellular responses to different signals including hypertonicity. The signaling response to hypertonicity is conserved across evolution from yeast to mammals in that it involves activation of p38/SAPK. However, very little is known about which upstream protein kinases mediate activation of p38 by hypertonicity in mammals. The MKKKs, MEKK3 and MEKK4, are upstream regulators of p38 in many cells. To investigate these signaling proteins as potential activators of p38 in the hypertonicity response, we generated stably transfected MDCK cells that express activated versions of MEKK3 or MEKK4, utilized RNA interference to deplete MEKK3, and employed pharmacological inhibition of p38 kinase. MEKK3-transfected cells demonstrated increased betaine transporter (BGT1) mRNA levels and upregulated tonicity enhancer (TonE)-driven luciferase activity under isotonic (basal) and hypertonic conditions compared with empty vector-transfected controls; small-interference RNA-mediated depletion of MEKK3 downregulated the activity of p38 kinase and decreased the expression of BGT1 mRNA. p38 Kinase inhibition abolished the effects of MEKK3 activation on BGT1 induction. In contrast, the response to hypertonicity in MEKK4-kA-transfected cells was similar to that observed in empty vector-transfected controls. Our data are consistent with the existence of an input from MEKK3 →→ p38 kinase →→ TonE. many organisms, including bacteria, yeast, plants, and animals, adapt to sustained hypertonic stress by the preferential accumulation of compatible organic osmolytes (56). In water-deprived mammals, for example, the extracellular osmolality of the kidney medulla may exceed 4,000 mosmol/kgH2O (38). Roughly one-half of the prevailing medullary interstitial solutes consist of urea, whereas the other half is composed of NaCl (15). Urea easily equilibrates across biological membranes and does not cause water shift between the intracellular and extracellular compartments. However, NaCl remains confined to the extracellular space, owing to the action of the Na-K-ATPase. An increase in the extracellular concentration of NaCl contributes to dehydration of the intracellular milieu (hypertonic stress), and restoration of intracellular volume in hypertonically stressed kidney cells requires the induction of a group of genes that lead to the accumulation of organic osmolytes intracellulary [BGT1 for betaine transporter (54), SMIT for inositol transporter (55), taurine transporter (49), and the aldose reductase enzyme (AR), which catalyze the reduction of d-glucose to the organic solute sorbitol (3)]. The transcriptional “machinery” that drives the expression of these genes [SMIT, BGT1, taurine transporter, and AR] under hypertonic conditions is similar (13, 19, 36, 44) and involves interaction between the cis-element [tonicity enhancer (TonE) (36), also known as osmotic response element (ORE) (13); referred to herein as TonE] and transcription factor TonE binding protein [TonEBP; (37), also known also as ORE binding protein (OREBP) (24) as well as NFAT5 (34); referred to herein as TonEBP]. Activation of TonE-mediated gene expression by hypertonicity is not unique to kidney cells [Madin-Darby canine kidney (MDCK) (36); rabbit kidney papillary epithelial cells (PAP-HT25) (25); mouse inner medullar collecting duct cells (mIMCD) (48)], as it has been shown to occur in neurons (35), human liver-derived HepG2 (41), Chang liver, Cos-7, and HeLa cells (24). Deletion of the TonEBP gene in mice blocks the expression of TonE-mediated gene expression in the kidney medulla almost completely, as evidenced by the diminished expression of the BGT1, SMIT, and AR genes. Remarkably, mice lacking TonEBP show atrophy of the renal medulla, which contains smaller cells and displays increased apoptosis (33). While transcriptional control of hypertonicity-induced genes in mammalian cells is reasonably well characterized, the signaling pathways leading to TonE-mediated gene expression need further delineation. In yeast, the adaptation to osmotic stress is dependent on the p38 MAPK homolog high-osmolarity glycerol 1 (HOG1) (7). Similarly, the induction of TonE-mediated gene expression in mammalian cells is p38 kinase dependent (41, 46) but requires cooperative action of Fyn, the catalytic subunit of PKA and the DNA damage-inducible kinase ATM (reviewed in Ref. 47). While ERK and JNK are induced by hypertonicity, the significance of their activation is not clear, as JNK and ERK do not appear to have an effect on TonE-mediated gene expression (reviewed in Ref. 47). In the current experiments, we sought to determine upstream signaling molecules in the p38 kinase cascade that “drive” the expression of hypertonicity-induced genes (represented by the betaine transporter BGT1) and affect TonE-mediated gene expression in kidney cells. The activity of p38 kinase is dependent on MAPK kinases (MKKs) and their activators, the MAPK kinase kinases (MKKKs; see review in Ref. 52). MEK kinase 1 (MEKK1; 1 of the MKKKs) is linked to JNK activation, whereas MEKK2 is linked to JNK and ERK activation (reviewed in Ref. 27). On the other hand, MEKK3 may activate ERK, JNK, and p38, whereas MEKK4 may activate JNK and p38 kinase (27). Hence, we hypothesized that MEKK3 and/or MEKK4 are likely mediators of p38 kinase activation in kidney cells under hypertonic conditions. Our data are consistent with the existence of MEKK3 → → → p38 kinase input to drive TonE-mediated gene expression.
Let’s take a poll: Working towards a more equitable classroom
(2023) Davis, Ann M.; Ahmed, Shazia A.
Immediate feedback is a successful engagement strategy that promotes active learning and fosters a supportive, inclusive classroom, especially when used with low stakes assignments or to gauge transfer of knowledge (Epstein & Brosvic, 2002, Goldstein & Wallis, 2015). The traditional approach is for students to purchase a physical device or license through an educational technology provider. The cost of these traditional services has been increasing, adding to the financial burden for our students in the midst of an ongoing global economic recession. If you have worked tirelessly and jumped through many hoops to adopt an OER textbook in order to bring down the cost of education for your students, you may feel guilty about adding back the expense of these educational tools. An alternative approach is to use a free or low-cost mobile app for student participation. In this article, we will present an array of free apps that we have used in various courses, and discuss the benefits and limitations of each.
The DBL-1/TGF-β signaling pathway tailors behavioral and molecular host responses to a variety of bacteria in Caenorhabditis elegans
(2023-09-26) Madhu, Bhoomi; Lakdawala, Mohammed Farhan; Gumienny, Tina L.
Generating specific, robust protective responses to different bacteria is vital for animal survival. Here, we address the role of transforming growth factor β (TGF-β) member DBL-1 in regulating signature host defense responses in Caenorhabditis elegans to human opportunistic Gram-negative and Gram-positive pathogens. Canonical DBL-1 signaling is required to suppress avoidance behavior in response to Gram-negative, but not Gram-positive bacteria. We propose that in the absence of DBL-1, animals perceive some bacteria as more harmful. Animals activate DBL-1 pathway activity in response to Gram-negative bacteria and strongly repress it in response to select Gram-positive bacteria, demonstrating bacteria-responsive regulation of DBL-1 signaling. DBL-1 signaling differentially regulates expression of target innate immunity genes depending on the bacterial exposure. These findings highlight a central role for TGF-β in tailoring a suite of bacteria-specific host defenses.
Small-scale extraction of Caenorhabditis elegans genomic DNA
(JOVE, 2022) Madhu, Bhoomi; Lakdawala, Mohammed Farhan; Gumienny, Tina L.
Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCRbased applications.
Reduced bone morphogenic protein signaling along the gut–neuron axis by heat shock factor promotes longevity
(Wiley, 2022) Arneaud, Sonja L.B.; McClendon, Jacob; Tatge, Lexus; Watterson, Abigail; Zuurbier, Kielen R.; Madhu, Bhoomi; Gumienny, Tina L.; Douglas, Peter M.
Aging is a complex and highly regulated process of interwoven signaling mechanisms. As an ancient transcriptional regulator of thermal adaptation and protein homeostasis, the Heat Shock Factor, HSF-1, has evolved functions within the nervous system to control age progression; however, the molecular details and signaling dynamics by which HSF-1 modulates age across tissues remain unclear. Herein, we report a nonautonomous mode of age regulation by HSF-1 in the Caenorhabditis elegans nervous system that works through the bone morphogenic protein, BMP, signaling pathway to modulate membrane trafficking in peripheral tissues. In particular, HSF-1 represses the expression of the neuron-specific BMP ligand, DBL-1, and initiates a complementary negative feedback loop within the intestine. By reducing receipt of DBL-1 in the periphery, the SMAD transcriptional coactivator, SMA-3, represses the expression of critical membrane trafficking regulators including Rab GTPases involved in early (RAB-5), late (RAB-7), and recycling (RAB-11.1) endosomal dynamics and the BMP receptor binding protein, SMA-10. This reduces cell surface residency and steady-state levels of the type I BMP receptor, SMA-6, in the intestine and further dampens signal transmission to the periphery. Thus, the ability of HSF-1 to coordinate BMP signaling along the gut–brain axis is an important determinate in age progression.
Both acute and consecutive days of formoterol stimulation influence myogenic, mitochondrial, and myomir gene expression in human skeletal muscle cells
(MDPI, 2023) Gordon, Ryan A.; Zumbro, Emily L.; Guerin, Gena D.; Sokoloski, Matthew L.; Ben-Ezra, Vic; Brower, Christopher S.; Rigby, Rhett B.; Duplanty, Anthony A.
Skeletal muscle physiology is regulated by microRNA that are localized within skeletal muscle (myomiRs). This study investigated how the expression of myomiRs and genes regulating skeletal muscle mass and myogenesis are influenced in response to acute and consecutive days of exercise-related signaling using the exercise mimetic, formoterol, in vitro. Human skeletal muscle cells were proliferated and differentiated for 6 days. Experimental conditions included: (a) control, (b) acute formoterol stimulation (AFS), and (c) consecutive days of formoterol stimulation (CFS). For AFS, myotubes were treated with 30 nM of formoterol for three hours on day 6 of differentiation, and this was immediately followed by RNA extraction. For CFS, myotubes were treated with 30 nM of formoterol for three hours on two or three consecutive days, with RNA extracted immediately following the final three-hour formoterol treatment. We observed increased myomiR expression for both AFS and CFS. AFS appeared to promote myogenesis, but this effect was lost with CFS. Additionally, we observed increased expression of genes involved in metabolism, mitochondrial biogenesis, and muscle protein degradation in response to AFS. myomiR and gene expression appear to be sensitive to acute and long-term exercise-related stimuli, and this likely contributes to the regulation of skeletal muscle mass.
A fluorescence-based reporter of Arginyltransferase 1 (ATE1)
(Texas Society for Microscopy, 2020) Kasu, Yasar Arfat T.; Dasgupta, Rinki; Brower, Christopher S.
Arginyltransferase 1 (ATE1) is an enzyme that catalyzes the transfer of arginine onto protein fragments with acidic N-termini. This is an essential step in the degradation of these fragments by the N-degron pathway of the ubiquitin proteasome system. Previous studies have shown that arginylation is required for the removal of specific fragments associated neurodegeneration, and that the loss of ATE1 activity leads to neurological problems. Interestingly, reduced ATE1 activity was also associated with fat loss and resistance to diet-induced obesity. Thus, the modulation of ATE1 holds promise for treating these increasingly common human diseases. To this end, we synthesized a cell-based reporter that employs direct fluorescence to monitor ATE1 activity. Using confocal microscopy and immunoblot analysis, we show that this reporter provides a robust readout of ATE1 activity in vivo. This reporter will be useful in screening approaches aimed to identify modulators of ATE1, which may ultimately have therapeutic potential.
Sex differences in the amygdaloid projections to the ventrolateral periaqueductal gray and their activation during inflammatory pain in the rat
(Elsevier, 2022) Cantu, Daisy J.; Kaur, Sukhbir; Murphy, Anne Z,; Averitt, Dayna L.
Preclinical and clinical studies have reported sex differences in pain and analgesia. These differences may be linked to anatomical structures of the central nervous system pain modulatory circuitry, and/or hormonal milieu. The midbrain periaqueductal gray (PAG) is a critical brain region for descending inhibition of pain. The PAG projects to the rostral ventromedial medulla (RVM), which projects bilaterally to the spinal cord to inhibit pain. In addition to pain, this descending circuit (or pathway) can be engaged by endogenous opioids (i.e., endorphins) or exogenous opioids (i.e., morphine), and we have previously reported sex differences in the activation of this circuit during pain and analgesia. Forebrain structures, including the amygdala, project to and engage the PAG-RVM circuit during persistent inflammatory pain. However, there are limited studies in females detailing this amygdalar-PAG pathway and its involvement during persistent inflammatory pain. The objective of the present study was to delineate the neural projections from the amygdala to the PAG in male and female rats to determine if they are sexually distinct in their anatomical organization. We also examined the activation of this pathway by inflammatory pain and the co-localization of receptors for estrogen. Injection of the retrograde tracer fluorogold (FG) into the ventrolateral PAG (vlPAG) resulted in dense retrograde labeling in both the central amygdala (CeA) and medial amygdala (MeA). While the number of CeA-vlPAG neurons were comparable between the sexes, there were more MeA-vlPAG neurons in females. Inflammatory pain resulted in greater activation of the amygdala in males; however, females displayed higher Fos expression within CeA-vlPAG projection neurons. Females expressed higher ERα in the MeA and CeA and the same was true of the projection neurons. Together, these data indicate that although the MeA-vlPAG projections are denser in females, inflammatory pain does not significantly activate these projections. In contrast, inflammatory pain resulted in a greater activation of the CeA-vlPAG pathway in females. As females experience a greater number of chronic pain syndromes, the CeA-vlPAG pathway may play a facilitatory (and not inhibitory) role in pain modulation.
Estrogen modulation of the pronociceptive effects of serotonin on female rat trigeminal sensory neurons is timing dependent and dosage dependent and requires estrogen receptor alpha
(Lippincott, 2022) Kaur, Sukhbir; Hickman, Taylor M.; Lopez-Ramirez, Angela; McDonald, Hanna; Lockhart, Lauren M.; Darwish, Omar; Averitt, Dayna L.
The role of the major estrogen estradiol (E2) on orofacial pain conditions remains controversial with studies reporting both a pronociceptive and antinociceptive role of E2. E2 modulation of peripheral serotonergic activity may be one mechanism underlying the female prevalence of orofacial pain disorders. We recently reported that female rats in proestrus and estrus exhibit greater serotonin (5HT)-evoked orofacial nocifensive behaviors compared with diestrus and male rats. Further coexpression of 5HT2A receptor mRNA in nociceptive trigeminal sensory neurons that express transient receptor potential vanilloid 1 ion channels contributes to pain sensitization. E2 may exacerbate orofacial pain through 5HT-sensitive trigeminal nociceptors, but whether low or high E2 contributes to orofacial pain and by what mechanism remains unclear. We hypothesized that steady-state exposure to a proestrus level of E2 exacerbates 5HT-evoked orofacial nocifensive behaviors in female rats, explored the transcriptome of E2-treated female rats, and determined which E2 receptor contributes to sensitization of female trigeminal sensory neurons. We report that a diestrus level of E2 is protective against 5HT-evoked orofacial pain behaviors, which increase with increasing E2 concentrations, and that E2 differentially alters several pain genes in the trigeminal ganglia. Furthermore, E2 receptors coexpressed with 5HT2A and transient receptor potential vanilloid 1 and enhanced capsaicin-evoked signaling in the trigeminal ganglia through estrogen receptor α. Overall, our data indicate that low, but not high, physiological levels of E2 protect against orofacial pain, and we provide evidence that estrogen receptor α receptor activation, but not others, contributes to sensitization of nociceptive signaling in trigeminal sensory neurons.
Sigma-1 receptors and progesterone metabolizing enzymes in nociceptive sensory neurons of the female rat trigeminal ganglia: A neural substrate for the antinociceptive actions of progesterone
(SAGE Publications, 2022) Hornung, Rebecca S.; Raut, Namrata G.R.; Cantu, Daisy J.; Lockhart, Lauren M.; Averitt, Dayna L.
Orofacial pain disorders are predominately experienced by women. Progesterone, a major ovarian hormone, is neuroprotective and antinociceptive. We recently reported that progesterone attenuates estrogen-exacerbated orofacial pain behaviors, yet it remains unclear what anatomical substrate underlies progesterone’s activity in the trigeminal system. Progesterone has been reported to exert protective effects through actions at intracellular progesterone receptors (iPR), membrane-progesterone receptors (mPR), or sigma 1 receptors (Sig-1R). Of these, the iPR and Sig-1R have been reported to have a role in pain. Progesterone can also have antinociceptive effects through its metabolite, allopregnanolone. Two enzymes, 5α-reductase and 3α-hydroxysteroid dehydrogenase (3α-HSD), are required for the metabolism of progesterone to allopregnanolone. Both progesterone and allopregnanolone rapidly attenuate pain sensitivity, implicating action of either progesterone at Sig-1R and/or conversion to allopregnanolone which targets GABAA receptors. In the present study, we investigated whether Sig-1 Rs are expressed in nociceptors within the trigeminal ganglia of cycling female rats and whether the two enzymes required for progesterone metabolism to allopregnanolone, 5α-reductase and 3α-hydroxysteroid dehydrogenase, are also present. Adult female rats from each stage of the estrous cycle were rapidly decapitated and the trigeminal ganglia collected. Trigeminal ganglia were processed by either fluorescent immunochemistry or western blotting to for visualization and quantification of Sig-1R, 5α-reductase, and 3α-hydroxysteroid dehydrogenase. Here we report that Sig-1Rs and both enzymes involved in progesterone metabolism are highly expressed in a variety of nociceptive sensory neuron populations in the female rat trigeminal ganglia at similar levels across the four stages of the estrous cycle. These data indicate that trigeminal sensory neurons are an anatomical substrate for the reported antinociceptive activity of progesterone via Sig-1R and/or conversion to allopregnanolone.
The effects of nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2) activation in preclinical models of peripheral neuropathic pain
(MDPI, 2022) Basu, Paramita; Averitt, Dayna L.; Maier, Camelia; Basu, Arpita
Oxidative stress, resulting from an imbalance between the formation of damaging free radicals and availability of protective antioxidants, can contribute to peripheral neuropathic pain conditions. Reactive oxygen and nitrogen species, as well as products of the mitochondrial metabolism such as superoxide anions, hydrogen peroxide, and hydroxyl radicals, are common free radicals. Nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is a transcription factor encoded by the NFE2L2 gene and is a member of the cap ‘n’ collar subfamily of basic region leucine zipper transcription factors. Under normal physiological conditions, Nrf2 remains bound to Kelch-like ECH-associated protein 1 in the cytoplasm that ultimately leads to proteasomal degradation. During peripheral neuropathy, Nrf2 can translocate to the nucleus, where it heterodimerizes with muscle aponeurosis fibromatosis proteins and binds to antioxidant response elements (AREs). It is becoming increasingly clear that the Nrf2 interaction with ARE leads to the transcription of several antioxidative enzymes that can ameliorate neuropathy and neuropathic pain in rodent models. Current evidence indicates that the antinociceptive effects of Nrf2 occur via reducing oxidative stress, neuroinflammation, and mitochondrial dysfunction. Here, we will summarize the preclinical evidence supporting the role of Nrf2 signaling pathways and Nrf2 inducers in alleviating peripheral neuropathic pain.
Seven ways to help burned-out faculty
(Magna Publications, 2022) Ahmed, Shazia; Sahlin, Claire
Faculty burnout was present in higher education long before COVID-19 because of the high emotional investment teaching requires of faculty. The pandemic has further exacerbated faculty exhaustion and stress.
The elongin B ubiquitin homology domain
(Elsevier, 1999) Brower, Christopher S.; Shilatifard, Ali; Mather, Timothy; Kamura, Takumi; Takagi, Yuichiro; Haque, Dewan; Treharne, Annemarie; Foundling, Stephen I.; Conaway, Joan Weliky; Conaway, Ronald C.
Mammalian Elongin B is a 118-amino acid protein composed of an 84-amino acid amino-terminal ubiquitin-like domain and a 34-amino acid carboxyl-terminal tail. Elongin B is found in cells as a subunit of the heterodimeric Elongin BC complex, which was originally identified as a positive regulator of RNA polymerase II elongation factor Elongin A and subsequently as a component of the multiprotein von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling complexes. As part of our effort to understand how the Elongin BC complex regulates the activity of Elongin A, we are characterizing Elongin B functional domains. In this report, we show that the Elongin B ubiquitin-like domain is necessary and sufficient for interaction with Elongin C and for positive regulation of Elongin A transcriptional activity. In addition, by site-directed mutagenesis of the Elongin B ubiquitin-like domain, we identify a short Elongin B region that is important for its interaction with Elongin C. Finally, we observe that both the ubiquitin-like domain and carboxyl-terminal tail are conserved in Drosophila melanogaster and Caenorhabditis elegans Elongin B homologs that efficiently substitute for mammalian Elongin B in reconstitution of the transcriptionally active Elongin ABC complex, suggesting that the carboxyl-terminal tail performs an additional function not detected in our assays.
A molecular basis for stabilization of the von hippel-lindau (VHL) tumor suppressor protein by components of the VHL ubiquitin ligase
(Elsevier, 2002) Kamura, Takumi; Brower, Christopher S.; Conaway, Ronald C.; Conaway, Joan Weliky
The multiprotein von Hippel-Lindau (VHL) tumor suppressor (CBCVHL, Cul2-ElonginBC-VHL) and SCF (Skp1-Cul1/Cdc53-F-box protein) complexes are members of structurally related families of E3 ubiquitin ligases that use a heterodimeric module composed of a member of the Cullin protein family and the RING finger protein Rbx1 (ROC1/Hrt1) to activate ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. VHL and F-box proteins function as the substrate recruitment subunits of CBCVHLand SCF complexes, respectively. In cells, many F-box proteins are short lived and are proposed to be ubiquitylated by an autocatalytic mechanism and destroyed by the proteasome following assembly into SCF complexes. In contrast, the VHL protein is stabilized by interaction with the Elongin B and C subunits of CBCVHL in cells. In this report, we have presented direct biochemical evidence that unlike the F-box protein Cdc4, which is ubiquitylated in vitro by Cdc34 in the context of the SCF, the VHL protein is protected from Ubc5-catalyzed ubiquitylation following assembly into the CBCVHL complex. CBCVHL is continuously required for negative regulation of hypoxia-inducible transcription factors in normoxic cells and of SCF complexes, many of which function only transiently during the cell cycle or in response to cellular signals. Our findings provide a molecular basis for the different modes of cellular regulation of VHL and F-box proteins and are consistent with the known roles of CBCVHL.
A mammalian homolog of drosophila melanogaster transcriptional coactivator intersex is a subunit of the mammalian Mediator Complex
(Elsevier, 2003) Sato, Shigeo; Tomomori-Sato, Chieri; Banks, Charles A.S.; Parmely, Tari J.; Sorokina, Irina; Brower, Christopher S.; Conaway, Ronald C.; Conaway, Joan Weliky
The multiprotein Mediator complex is a coactivator required for transcriptional activation of RNA polymerase II transcribed genes by DNA binding transcription factors. We previously partially purified a Med8-containing Mediator complex from rat liver nuclei (Brower, C. S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353–10358). Analysis of proteins present in the most highly enriched Mediator fractions by tandem mass spectrometry led to the identification of several new mammalian Mediator subunits, as well as several potential Mediator subunits. Here we identify one of these proteins, encoded by the previously uncharacterized AK000411 open reading frame, as a new subunit of the mammalian Mediator complex. The AK000411 protein, which we designate hIntersex (human Intersex), shares significant sequence similarity with the Drosophila melanogaster intersex protein, which has functional properties expected of a transcriptional coactivator specific for the Drosophila doublesex transactivator. In addition, we show that hIntersex assembles into a subcomplex with Mediator subunits p28b and TRFP. Taken together, our findings identify a new subunit of the mammalian Mediator and shed new light on the architecture of the mammalian Mediator complex.
Identification of mammalian mediator subunits with similarities to yeast mediator subunits srb5, srb6, med11, and ROX3
(Elsevier, 2003) Sato, Shigeo; Tomomori-Sato, Chieri; Banks, Charles A.S.; Sorokina, Irina; Parmely, Tari J.; Kong, Stephanie E.; Jin, Jingji; Cai, Yong; Lane, William S.; Brower, Christopher S.; Conaway, Ronald C.; Conaway, Joan Weliky
The Mediator is a multiprotein coactivator required for activation of RNA polymerase II transcription by DNA binding transactivators. We recently identified a mammalian homologue of yeast Mediator subunit Med8 and partially purified a Med8-containing Mediator complex from rat liver nuclei (Brower, C. S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353–10358). Analysis of proteins present in the most highly purified Med8-containing fractions by tandem mass spectrometry led to the identification of many known mammalian Mediator subunits, as well as four potential Mediator subunits exhibiting sequence similarity to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3. Here we present direct biochemical evidence that these four proteins are bona fide mammalian Mediator subunits. In addition, we identify direct pairwise binding partners of these proteins among the known mammalian Mediator subunits. Taken together, our findings identify a collection of novel mammalian Mediator subunits and shed new light on the underlying architecture of the mammalian Mediator complex.
A mammalian mediator subunit that shares properties with saccharomyces cerevisiae mediator subunit CSE2
(Elsevier, 2004) Tomomori-Sato, Chieri; Sato, Shigeo; Parmely, Tari J.; Banks, Charles A.S.; Sorokina, Irina; Florens, Laurence; Zybailov, Boris; Washburn, Michael P.; Brower, Christopher S.; Conaway, Ronald C.; Conaway, Joan Weliky
The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Sorokina, I., Roeder, R.G., Conaway, J.W., and Conaway, R.C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353–10358). Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193 open reading frame. In this study, we present direct biochemical evidence that the FLJ10193 protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex. In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog. Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex.
Ablation of arginylation in the mouse N-end rule pathway: Loss of fat, higher metabolic rate, damaged spermatogenesis, and neurological perturbations
(Public Library of Science, 2009) Brower, Christopher S.; Varshavsky, Alexander
In the N-end rule pathway of protein degradation, the destabilizing activity of N-terminal Asp, Glu or (oxidized) Cys residues requires their conjugation to Arg, which is recognized directly by pathway’s ubiquitin ligases. N-terminal arginylation is mediated by the Ate1 arginyltransferase, whose physiological substrates include the Rgs4, Rgs5 and Rgs16 regulators of G proteins. Here, we employed the Cre-lox technique to uncover new physiological functions of N-terminal arginylation in adult mice. We show that postnatal deletion of mouse Ate1 (its unconditional deletion is embryonic lethal) causes a rapid decrease of body weight and results in early death of ,15% of Ate1-deficient mice. Despite being hyperphagic, the surviving Ate1-deficient mice contain little visceral fat. They also exhibit an increased metabolic rate, ectopic induction of the Ucp1 uncoupling protein in white fat, and are resistant to diet-induced obesity. In addition, Ate1-deficient mice have enlarged brains, an enhanced startle response, are strikingly hyperkinetic, and are prone to seizures and kyphosis. Ate1- deficient males are also infertile, owing to defects in Ate12/2 spermatocytes. The remarkably broad range of specific biological processes that are shown here to be perturbed by the loss of N-terminal arginylation will make possible the dissection of regulatory circuits that involve Ate1 and either its known substrates, such as Rgs4, Rgs5 and Rgs16, or those currently unknown.

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