Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology

Pharmacological inhibitors of cysteine proteases have provideduseful insights into the regulation of calpain activity inerythrocytes. However, the precise biological function of calpainactivity in erythrocytes remains poorly understood. Erythrocytesexpress calpain-1, an isoform regulated by calpastatin, theendogenous inhibitor of calpains. In the present study, weinvestigated the function of calpain-1 in mature erythrocytes usingour calpain-1-null [KO (knockout)] mouse model. The calpain-1gene deletion results in improved erythrocyte deformabilitywithout any measurable effect on erythrocyte lifespan in vivo.The calcium-induced sphero-echinocyte shape transition iscompromised in the KO erythrocytes. Erythrocyte membraneproteins ankyrin, band 3, protein 4.1R, adducin and dematin aredegraded in the calcium-loaded normal erythrocytes but not inthe KO erythrocytes. In contrast, the integrity of spectrin andits state of phosphorylation are not affected in the calciumloadederythrocytes of either genotype. To assess the functionalconsequences of attenuated cytoskeletal remodelling in the KOerythrocytes, the activity of major membrane transporters wasmeasured. The activity of the K+\u2013Cl 12 co-transporter and theGardos channel was significantly reduced in the KO erythrocytes.Similarly, the basal activity of the calcium pump was reducedin the absence of calmodulin in the KO erythrocyte membrane.Interestingly, the calmodulin-stimulated calcium pump activitywas significantly elevated in the KO erythrocytes, implying awider range of pump regulation by calcium and calmodulin. Takentogether, and with the atomic force microscopy of the skeletalnetwork, the results of the present study provide the first evidencefor the physiological function of calpain-1 in erythrocytes withtherapeutic implications for calcium imbalance pathologies suchas sickle cell disease

Pharmacological inhibition of calpain-1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease

ABSTRACT Sickle cell disease (SCD) is a globallydistributed hereditary red blood cell (RBC) disorder.One of the hallmarks of SCD is the presence ofcirculating dense RBCs, which are important in SCDrelatedclinical manifestations. In human dense sicklecells, we found reduced calpastatin activity and proteinexpression compared to either healthy RBCs or unfractionatedsickle cells, suggesting an imbalance betweenactivator and inhibitor of calpain-1 in favor of activatorin dense sickle cells. Calpain-1 is a nonlysosomal cysteineproteinase that modulates multiple cell functionsthrough the selective cleavage of proteins. To investigatethe relevance of this observation in vivo, weevaluated the effects of the orally active inhibitor ofcalpain-1, BDA-410 (30 mg/kg/d), on RBCs from SADmice, a mouse model for SCD. In SAD mice, BDA-410improved RBC morphology, reduced RBC density (D20;from 11060.001 to 11000.001 g/ml; P<0.05) andincreased RBC-K content (from 36410 to 42912.3mmol/kg Hb; P<0.05), markedly reduced the activityof the Ca2-activated Kchannel (Gardos channel), anddecreased membrane association of peroxiredoxin-2.The inhibitory effect of calphostin C, a specific inhibitorof protein kinase C (PKC), on the Gardos channelwas eliminated after BDA-410 treatment, which suggeststhat calpain-1 inhibition affects the PKC-dependentfraction of the Gardos channel. BDA-410 preventedhypoxia-induced RBC dehydration and K lossin SAD mice. These data suggest a potential role ofBDA-410 as a novel therapeutic agent for treatment ofSCD

PAR2 Pepducin-Based Suppression of Inflammation and Itch in Atopic Dermatitis Models

We thank Joe Stevens for assistance in conducting dermatitis models. This study was funded in whole or in part by AR067617 (AK, LC) from the US National Institute of Arthritis and Musculoskeletal and Skin Diseases.PAR2 has been proposed to contribute to lesion formation and intense itch in atopic dermatitis. Here, we tested the ability of a cell-penetrating pepducin, PZ-235, to mitigate the potentially deleterious effects of PAR2 in models of atopic dermatitis. PZ-235 significantly inhibited PAR2-mediated expression of inflammatory factors NF-κB, TSLP, TNF-α and differentiation marker K10 by 94%-98% (P < 0.001) in human keratinocytes and suppressed IL-4 and IL-13 by 68%-83% (P < 0.05) in mast cells. In delayed pepducin treatment models of oxazolone- and DNFB-induced dermatitis, PZ-235 significantly attenuated skin thickening by 43%-100% (P < 0.01) and leukocyte crusting by 57% (P < 0.05), and it inhibited ex vivo chemotaxis of leukocytes toward PAR2 agonists. Daily PZ-235 treatment of filaggrin-deficient mice exposed to dust mite allergens for 8 weeks significantly suppressed total leukocyte and T-cell infiltration by 50%-68%; epidermal thickness by 60%-77%; and skin thickening, scaling, excoriation, and total lesion severity score by 46%-56%. PZ-235 significantly reduced itching caused by wasp venom peptide degranulation of mast cells in mice by 51% (P < 0.05), which was comparable to the protective effects conferred by PAR2 deficiency. Taken together, these results suggest that a PAR2 pepducin may confer broad therapeutic benefits as a disease-modifying treatment for atopic dermatitis and itch

Dual Mechanism of Integrin alpha(IIb)beta(3) Closure in Procoagulant Platelets

Aggregation of platelets via activated integrin α(IIb)β(3) is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that α(IIb)β(3) inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the β(3) chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed α(IIb)β(3) inactivation. Integrin α(IIb)β(3) inactivation was unchanged in platelets from Capn1(−/−) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced α(IIb)β(3) inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(−/−) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and α(IIb)β(3) inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of α(IIb)β(3) inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation

Targeted Gene Inactivation of Calpain-1 Suppresses Cortical Degeneration Due to Traumatic Brain Injury and Neuronal Apoptosis Induced by Oxidative Stress*

Calpains are calcium-regulated cysteine proteases that have been implicated in the regulation of cell death pathways. Here, we used our calpain-1 null mouse model to evaluate the function of calpain-1 in neural degeneration following a rodent model of traumatic brain injury. In vivo, calpain-1 null mice show significantly less neural degeneration and apoptosis and a smaller contusion 3 days post-injury than wild type littermates. Protection from traumatic brain injury corroborated with the resistance of calpain-1 neurons to apoptosis induced by oxidative stress. Biochemical analysis revealed that caspase-3 activation, extracellular calcium entry, mitochondrial membrane permeability, and release of apoptosis-inducing factor from mitochondria are partially blocked in the calpain-1 null neurons. These findings suggest that the calpain-1 knock-out mice may serve as a useful model system for neuronal protection and apoptosis in traumatic brain injury and other neurodegenerative disorders in which oxidative stress plays a role