Acute PDE4 Inhibition Induces a Transient Increase in Blood Glucose in Mice.

cAMP-phosphodiesterase 4 (PDE4) inhibitors are currently approved for the treatment of inflammatory diseases. There is interest in expanding the therapeutic application of PDE4 inhibitors to metabolic disorders, as their chronic application induces weight loss in patients and animals and improves glucose handling in mouse models of obesity and diabetes. Unexpectedly, we have found that acute PDE4 inhibitor treatment induces a temporary increase, rather than a decrease, in blood glucose levels in mice. Blood glucose levels in postprandial mice increase rapidly upon drug injection, reaching a maximum after ~45 min, and returning to baseline within ~4 h. This transient blood glucose spike is replicated by several structurally distinct PDE4 inhibitors, suggesting that it is a class effect of PDE4 inhibitors. PDE4 inhibitor treatment does not reduce serum insulin levels, and the subsequent injection of insulin potently reduces PDE4 inhibitor-induced blood glucose levels, suggesting that the glycemic effects of PDE4 inhibition are independent of changes in insulin secretion and/or sensitivity. Conversely, PDE4 inhibitors induce a rapid reduction in skeletal muscle glycogen levels and potently inhibit the uptake of 2-deoxyglucose into muscle tissues. This suggests that reduced glucose uptake into muscle tissue is a significant contributor to the transient glycemic effects of PDE4 inhibitors in mice.

Assessment of PDE4 Inhibitor-Induced Hypothermia as a Correlate of Nausea in Mice.

Treatment with PAN-PDE4 inhibitors has been shown to produce hypothermia in multiple species. Given the growing body of evidence that links nausea and emesis to disturbances in thermoregulation in mammals, we explored PDE4 inhibitor-induced hypothermia as a novel correlate of nausea in mice. Using knockout mice for each of the four PDE4 subtypes, we show that selective inactivation of individual PDE4 subtypes per se does not produce hypothermia, which must instead require the concurrent inactivation of multiple (at least two) PDE4 subtypes. These findings contrast with the role of PDE4s in shortening the duration of α2-adrenoceptor-dependent anesthesia, a behavioral surrogate previously used to assess the emetic potential of PDE4 inhibitors, which is exclusively affected by inactivation of PDE4D. These different outcomes are rooted in the distinct molecular mechanisms that drive these two paradigms; acting as a physiologic α2-adrenoceptor antagonist produces the effect of PDE4/PDE4D inactivation on the duration of α2-adrenoceptor-dependent anesthesia, but does not mediate the effect of PDE4 inhibitors on body temperature in mice. Taken together, our findings suggest that selective inhibition of any individual PDE4 subtype, including inhibition of PDE4D, may be free of nausea and emesis.

Ablation of PDE4B protects from Pseudomonas aeruginosa-induced acute lung injury in mice by ameliorating the cytostorm and associated hypothermia.

Pseudomonas aeruginosa is a frequent cause of hospital-acquired lung infections characterized by hyperinflammation, antibiotic resistance, and high morbidity/mortality. Here, we show that the genetic ablation of one cAMP-phosphodiesterase 4 subtype, PDE4B, is sufficient to protect mice from acute lung injury induced by P aeruginosa infection as it reduces pulmonary and systemic levels of pro-inflammatory cytokines, as well as pulmonary vascular leakage and mortality. Surprisingly, despite dampening immune responses, bacterial clearance in the lungs of PDE4B-KO mice is significantly improved compared to WT controls. In wildtypes, P aeruginosa-infection produces high systemic levels of several cytokines, including TNF-α, IL-1ß, and IL-6, that act as cryogens and render the animals hypothermic. This, in turn, diminishes their ability to clear the bacteria. Ablation of PDE4B curbs both the initial production of acute response cytokines, including TNF-α and IL-1ß, as well as their downstream signaling, specifically the induction of the secondary-response cytokine IL-6. This synergistic action protects PDE4B-KO mice from the deleterious effects of the P aeruginosa-induced cytostorm, while concurrently improving bacterial clearance, rather than being immunosuppressive. These benefits of PDE4B ablation are in contrast to the effects resulting from treatment with PAN-PDE4 inhibitors, which have been shown to increase bacterial burden and dissemination. Thus, PDE4B represents a promising therapeutic target in settings of P aeruginosa lung infections.

The cAMP-phosphodiesterase 4 (PDE4) controls ß-adrenoceptor- and CFTR-dependent saliva secretion in mice.

Saliva, while often taken for granted, is indispensable for oral health and overall well-being, as inferred from the significant impairments suffered by patients with salivary gland dysfunction. Here, we show that treatment with several structurally distinct PAN-PDE4 inhibitors, but not a PDE3 inhibitor, induces saliva secretion in mice, indicating it is a class-effect of PDE4 inhibitors. In anesthetized mice, while neuronal regulations are suppressed, PDE4 inhibition potentiates a ß-adrenoceptor-induced salivation, that is ablated by the ß-blocker Propranolol and is absent from homozygous ΔF508-CFTR mice lacking functional CFTR. These data suggest that PDE4 acts within salivary glands to gate saliva secretion that is contingent upon the cAMP/PKA-dependent activation of CFTR. Indeed, PDE4 contributes the majority of total cAMP-hydrolytic capacity in submandibular-, sublingual-, and parotid glands, the three major salivary glands of the mouse. In awake mice, PDE4 inhibitor-induced salivation is reduced by CFTR deficiency or ß-blockers, but also by the muscarinic blocker Atropine, suggesting an additional, central/neuronal mechanism of PDE4 inhibitor action. The PDE4 family comprises four subtypes, PDE4A-D. Ablation of PDE4D, but not PDE4A-C, produced a minor effect on saliva secretion, implying that while PDE4D may play a predominant role, PDE4 inhibitor-induced salivation results from the concurrent inactivation of multiple (at least two) PDE4 subtypes. Taken together, our data reveal a critical role for PDE4/PDE4D in controlling CFTR function in an in vivo model and in inducing salivation, hinting at a therapeutic potential of PDE4 inhibition for cystic fibrosis and conditions associated with xerostomia.

Inhibition of cAMP-phosphodiesterase 4 (PDE4) potentiates the anesthetic effects of Isoflurane in mice.

Despite major advances, there remains a need for novel anesthetic drugs or drug combinations with improved efficacy and safety profiles. Here, we show that inhibition of cAMP-phosphodiesterase 4 (PDE4), while not inducing anesthesia by itself, potently enhances the anesthetic effects of Isoflurane in mice. Treatment with several distinct PAN-PDE4 inhibitors, including Rolipram, Piclamilast, Roflumilast, and RS25344, significantly delayed the time-to-righting after Isoflurane anesthesia. Conversely, treatment with a PDE3 inhibitor, Cilostamide, or treatment with the potent, but non-brain-penetrant PDE4 inhibitor YM976, had no effect. These findings suggest that potentiation of Isoflurane hypnosis is a class effect of brain-penetrant PDE4 inhibitors, and that they act by synergizing with Isoflurane in inhibiting neuronal activity. The PDE4 family comprises four PDE4 subtypes, PDE4A to PDE4D. Genetic deletion of any of the four PDE4 subtypes in mice did not affect Isoflurane anesthesia per se. However, PDE4D knockout mice are largely protected from the effect of pharmacologic PDE4 inhibition, suggesting that PDE4D is the predominant, but not the sole PDE4 subtype involved in potentiating Isoflurane anesthesia. Pretreatment with Naloxone or Propranolol alleviated the potentiating effect of PDE4 inhibition, implicating opioid- and ß-adrenoceptor signaling in mediating PDE4 inhibitor-induced augmentation of Isoflurane anesthesia. Conversely, stimulation or blockade of α1-adrenergic, α2-adrenergic or serotonergic signaling did not affect the potentiation of Isoflurane hypnosis by PDE4 inhibition. We further show that pretreatment with a PDE4 inhibitor boosts the delivery of bacteria into the lungs of mice after intranasal infection under Isoflurane, thus providing a first example that PDE4 inhibitor-induced potentiation of Isoflurane anesthesia can critically impact animal models and must be considered as a factor in experimental design. Our findings suggest that PDE4/PDE4D inhibition may serve as a tool to delineate the exact molecular mechanisms of Isoflurane anesthesia, which remain poorly understood, and may potentially be exploited to reduce the clinical doses of Isoflurane required to maintain hypnosis.

PAN-selective inhibition of cAMP-phosphodiesterase 4 (PDE4) induces gastroparesis in mice.

Inhibitors of cAMP-phosphodiesterase 4 (PDE4) exert a number of promising therapeutic benefits, but adverse effects, in particular emesis and nausea, have curbed their clinical utility. Here, we show that PAN-selective inhibition of PDE4, but not inhibition of PDE3, causes a time- and dose-dependent accumulation of chow in the stomachs of mice fed ad libitum without changing the animals' food intake or the weight of their intestines, suggesting that PDE4 inhibition impairs gastric emptying. Indeed, PDE4 inhibition induced gastric retention in an acute model of gastric motility that traces the passage of a food bolus through the stomach over a 30 minutes time period. In humans, abnormal gastric retention of food is known as gastroparesis, a syndrome predominated by nausea (>90% of cases) and vomiting (>80% of cases). We thus explored the abnormal gastric retention induced by PDE4 inhibition in mice under the premise that it may represent a useful correlate of emesis and nausea. Delayed gastric emptying was produced by structurally distinct PAN-PDE4 inhibitors including Rolipram, Piclamilast, Roflumilast, and RS25344, suggesting that it is a class effect. PDE4 inhibitors induced gastric retention at similar or below doses commonly used to induce therapeutic benefits (e.g., 0.04 mg/kg Rolipram), thus mirroring the narrow therapeutic window of PDE4 inhibitors in humans. YM976, a PAN-PDE4 inhibitor that does not efficiently cross the blood-brain barrier, induced gastroparesis only at significantly higher doses (≥1 mg/kg). This suggests that PDE4 inhibition may act in part through effects on the autonomic nervous system regulation of gastric emptying and that PDE4 inhibitors that are not brain-penetrant may have an improved safety profile. The PDE4 family comprises four subtypes, PDE4A, B, C, and D. Selective ablation of any of these subtypes in mice did not induce gastroparesis per se, nor did it protect from PAN-PDE4 inhibitor-induced gastroparesis, indicating that gastric retention may result from the concurrent inhibition of multiple PDE4s. Thus, potentially, any of the four PDE4 subtypes may be targeted individually for therapeutic benefits without inducing nausea or emesis. Acute gastric retention induced by PDE4 inhibition is alleviated by treatment with the widely used prokinetic Metoclopramide, suggesting a potential of this drug to alleviate the side effects of PDE4 inhibitors. Finally, given that the cause of gastroparesis remains largely idiopathic, our findings open the possibility that a physiologic or pathophysiologic downregulation of PDE4 activity/expression may be causative in a subset of patients.

Inhibition of type 4 cAMP-phosphodiesterases (PDE4s) in mice induces hypothermia via effects on behavioral and central autonomous thermoregulation.

Inhibitors of Type 4 cAMP-phosphodiesterases (PDE4s) exert a number of promising therapeutic benefits, including potent anti-inflammatory, memory- and cognition-enhancing, metabolic, and antineoplastic effects. We report here that treatment with a number of distinct PDE4 inhibitors, including Rolipram, Piclamilast, Roflumilast and RS25344, but not treatment with the PDE3-selective inhibitor Cilostamide, induces a rapid (10-30 min), substantial (-5 °C) and long-lasting (up to 5 h) decrease in core body temperature of C57BL/6 mice; thus, identifying a critical role of PDE4 also in the regulation of body temperature. As little as 0.04 mg/kg of the archetypal PDE4 inhibitor Rolipram induces hypothermia. As similar or higher doses of Rolipram were used in a majority of published animal studies, most of the reported findings are likely paralleled by, or potentially impacted by hypothermia induced by these drugs. We further show that PDE4 inhibition affects central body temperature regulation and acts by lowering the cold-defense balance point of behavioral (including posture and locomotion) and autonomous (including cutaneous tail vasodilation) cold-defense mechanisms. In line with the idea of an effect on central body temperature regulation, hypothermia is induced by moderate doses of various brain-penetrant PDE4 inhibitors, but not by similar doses of YM976, a PDE4 inhibitor that does not efficiently cross the blood-brain barrier. Finally, to begin delineating the mechanism of drug-induced hypothermia, we show that blockade of D2/3-type dopaminergic, but not ß-adrenergic, H1-histaminergic or opiate receptors, can alleviate PDE4 inhibitor-induced hypothermia. We thus propose that increased D2/3-type dopaminergic signaling, triggered by PDE4 inhibitor-induced and cAMP-mediated dopamine release in the thermoregulatory centers of the hypothalamus, is a significant contributor to PDE4 inhibitor-induced hypothermia.

The specialized unfolded protein response of B lymphocytes: ATF6α-independent development of antibody-secreting B cells.

B lymphocytes, like all mammalian cells, are equipped with the unfolded protein response (UPR), a complex signaling system allowing for both pro- and mal-adaptive responses to increased demands on the endoplasmic reticulum (ER). The UPR is comprised of three signaling pathways initiated by the ER transmembrane stress sensors, IRE1α/ß, PERK and ATF6α/ß. Activation of IRE1 yields XBP1(S), a transcription factor that directs expansion of the ER and enhances protein biosynthetic and secretory machinery. XBP1(S) is essential for the differentiation of B lymphocytes into antibody-secreting cells. In contrast, the PERK pathway, a regulator of translation and transcription, is dispensable for the generation of antibody-secreting cells. Functioning as a transcription factor, ATF6α can augment ER quality control processes and drive ER expansion, but the potential role of this UPR pathway in activated B cells has not been investigated. Here, we report studies of ATF6α-deficient B cells demonstrating that ATF6α is not required for the development of antibody-secreting cells. Thus, when B cells are stimulated to secrete antibody, a specialized UPR relies exclusively on the IRE1-XBP1 pathway to remodel the ER and expand cellular secretory capacity.

MicroRNA-30c-2* limits expression of proadaptive factor XBP1 in the unfolded protein response.

Stress in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a multifaceted signaling system coordinating translational control and gene transcription to promote cellular adaptation and survival. Microribonucleic acids (RNAs; miRNAs), single-stranded RNAs that typically function as posttranscriptional modulators of gene activity, have been shown to inhibit translation of certain secretory pathway proteins during the UPR. However, it remains unclear whether miRNAs regulate UPR signaling effectors directly. In this paper, we report that a star strand miRNA, miR-30c-2* (recently designated miR-30c-2-3p), is induced by the protein kinase RNA activated-like ER kinase (PERK) pathway of the UPR and governs expression of XBP1 (X-box binding protein 1), a key transcription factor that augments secretory capacity and promotes cell survival in the adaptive UPR. These data provide the first link between an miRNA and direct regulation of the ER stress response and reveal a novel molecular mechanism by which the PERK pathway, via miR-30c-2*, influences the scale of XBP1-mediated gene expression and cell fate in the UPR.

Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer.

Ser/Thr protein phosphatase 5 (PP5) regulates several signaling-cascades that suppress growth and/or facilitate apoptosis in response to genomic stress. The expression of PP5 is responsive to hypoxia inducible factor-1 (HIF-1) and estrogen, which have both been linked to the progression of human breast cancer. Still, it is not clear if PP5 plays a role in the development of human cancer. Here, immunostaining of breast cancer tissue-microarrays (TMAs) revealed a positive correlation between PP5 over-expression and ductal carcinoma in situ (DCIS; P value 0.0028), invasive ductal carcinoma (IDC; P value 0.012) and IDC with metastases at the time of diagnosis (P value 0.0001). In a mouse xenograft model, the constitutive over-expression of PP5 was associated with an increase in the rate of tumor growth. In a MCF-7 cell culture model over-expression correlated with both an increase in the rate of proliferation and protection from cell death induced by oxidative stress, UVC-irradiation, adriamycin, and vinblastine. PP5 over-expression had no apparent effect on the sensitivity of MCF-7 cells to taxol or rapamycin. Western analysis of extracts from cells over-expressing PP5 revealed a decrease in the phosphorylation of known substrates for PP5. Together, these studies indicate that elevated levels of PP5 protein occur in human breast cancer and suggest that PP5 over-expression may aid tumor progression.

Cantharidin-induced mitotic arrest is associated with the formation of aberrant mitotic spindles and lagging chromosomes resulting, in part, from the suppression of PP2Aalpha.

Cantharidin, a natural vesicant, inhibits the activity of several PPP family phosphatases, displays antitumor activity, and induces apoptosis in many types of tumor cells. However, the molecular mechanisms underlying the antitumor activity of cantharidin are not clear. Here, dose-response studies confirm a strong correlation between the suppression of phosphatase activity and cell death. Flow cytometry analysis indicates that before apoptosis, cantharidin delays cell cycle progression following DNA replication with no apparent effect on G(1)-S or S-G(2) phase progression. In contrast, studies with double thymidine-synchronized populations of cells indicate that cantharidin can rapidly arrest growth when added during G(2) or early M phase. Immunostaining indicates that cell cycle arrest occurs before the completion of mitosis and is associated with the appearance of aberrant mitotic spindles. Live cell imaging with time-lapse microscopy shows that cantharidin disrupts the metaphase alignment of chromosomes and produces a prolonged mitotic arrest, with the onset of apoptosis occurring before the onset of anaphase. To explore the contribution of individual phosphatases, antisense oligonucleotides and small interfering RNA were developed to suppress the expression of cantharidin-sensitive phosphatases. The suppression of PP2Aalpha, but not PP2Abeta, is sufficient to induce metaphase arrest, during which time lagging chromosomes are observed moving between the spindle poles and the metaphase plate. Immunostaining revealed slightly abnormal, yet predominately bipolar, mitotic spindles. Nonetheless, after a 10- to 15-hour delay, the cells enter anaphase, suggesting that an additional cantharidin-sensitive phosphatase is involved in the progression from metaphase into anaphase or to prevent the onset of apoptosis in cells arrested during mitosis.

Constitutive over expression of serine/threonine protein phosphatase 5 (PP5) augments estrogen-dependent tumor growth in mice.

Serine/threonine protein phosphatase 5 (PP5) appears to play an underappreciated role in the regulation of cellular proliferation. In estrogen-responsive cells, PP5 expression is stimulated by 17 beta-estradiol, and in a variety of p53 wild-type tumor cells the suppression of PP5 expression with ISIS 15534 inhibits growth. To further explore the relationship between PP5 and the development of human cancer, here we tested the effect of elevated PP5 expression on tumor growth using a mouse xenograph model and a stable MCF-7 cell line in which the expression of wild-type PP5 was placed under the control of tetracycline-off regulated transactivator and operator plasmids. In the xenograph model a modest two fold increase in PP5 protein levels significantly enhanced the growth rate of estrogen-dependent tumors, suggesting PP5 plays a positive role in tumor development.

Ser/Thr protein phosphatase 5 inactivates hypoxia-induced activation of an apoptosis signal-regulating kinase 1/MKK-4/JNK signaling cascade.

Mitogen-activated protein kinase (MAPK) signaling cascades are multifunctional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. Since the activation/propagation of MAPK signaling requires the sequential phosphorylation of many downstream proteins, the phosphatases that dephosphorylate MAPKs represent critical elements in the control of MAPK-signaling networks. Here we show that hypoxia induces a transient increase in the activity of apoptosis signal-regulating kinase 1 (ASK-1), a MAPKKK that responds to oxidative stress by triggering cascades leading to the phosphorylation/activation of c-Jun N-terminal kinases (JNK) and p38-MAPK. Hypoxia-induced ASK-1/MKK-4/JNK signaling is suppressed by serine/threonine protein phosphatase type 5 (PP5), which acts to turn off ASK-1/MKK-4/JNK signaling via two mechanisms. First, in a rapid response hypoxia facilitates the association of endogenous PP5 with ASK-1. PP5 binds to the C-terminal domain of ASK-1, and studies with siRNA targeting PP5 indicate that PP5 acts to suppress the phosphorylation of MKK4 (Thr-261), JNK (Thr-183/Tyr-185), and c-Jun (Ser-63) without affecting the activating phosphorylation of p38 MAPK (Thr-180/Tyr-182), p44/p42-MAPK/ERK1/2 (Thr-202/Tyr-204), or c-Jun protein levels. If hypoxia is prolonged, the expression of PP5 is increased due to the activation of a transcriptional activator, which was identified as hypoxia-inducible factor-1. Together, these studies indicate that PP5 plays an important role in the survival of cells in a low oxygen environment by suppressing a hypoxia-induced ASK-1/MKK4/JNK signaling cascade that promotes an apoptotic response.

Identification of a functional link for the p53 tumor suppressor protein in dexamethasone-induced growth suppression.

Serine/threonine phosphatase 5 (PP5) can act as a suppresser of p53-dependent growth suppression and has been reported to associate with several proteins, including the glucocorticoid receptor/heat-shock protein-90 complex. Still, the physiological/pathological roles of PP5 are unclear. To characterize the relationship of PP5, glucocorticoid receptor activation and p53, here we describe the development of chimeric antisense oligonucleotides that potently inhibit human p53 expression. This allowed us to regulate the expression of either p53 (e.g. with ISIS 110332) or PP5 (e.g. with ISIS 15534) in genetically identical cells. Studies with ISIS 110332 revealed that the suppression of p53 expression is associated with a decrease in the basal expression of the cyclin-dependent kinase inhibitor protein, p21(WAF1/Cip1), and a concomitant increase in the rate of cell proliferation. Suppression of p53 also blocks dexamethasone-induced p21(WAF1/Cip1) expression and G(1)-growth arrest. Furthermore, treatment with ISIS 110332, but not the mismatched controls, ablates the suppression of growth produced by prior treatment with dexamethasone. Additional studies revealed that dexamethasone-dependent p21(WAF1/Cip1) expression occurs without an apparent change in p53 protein levels or the phosphorylation status of p53 at Ser-6, -37, or -392. However, dexamethasone treatment is associated with an increase in p53 phosphorylation at Ser-15. Suppression of PP5 expression with ISIS 15534 also results in the hyperphosphorylation of p53 at Ser-15. Together, these findings indicate that the basal expression of p53 plays a functional role in a glucocorticoid receptor-mediated response regulating the expression of p21(Waf1/Cip1) via a mechanism that is suppressed by PP5 and associated with the phosphorylation of p53 at Ser-15.