iPSC-Derived Endothelial Cells Affect Vascular Function in a Tissue-Engineered Blood Vessel Model of Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder caused by a point mutation in the Lamin A gene that produces the protein progerin. Progerin toxicity leads to accelerated aging and death from cardiovascular disease. To elucidate the effects of progerin on endothelial cells, we prepared tissue-engineered blood vessels (viTEBVs) using induced pluripotent stem cell-derived smooth muscle cells (viSMCs) and endothelial cells (viECs) from HGPS patients. HGPS viECs aligned with flow but exhibited reduced flow-responsive gene expression and altered NOS3 levels. Relative to viTEBVs with healthy cells, HGPS viTEBVs showed reduced function and exhibited markers of cardiovascular disease associated with endothelium. HGPS viTEBVs exhibited a reduction in both vasoconstriction and vasodilation. Preparing viTEBVs with HGPS viECs and healthy viSMCs only reduced vasodilation. Furthermore, HGPS viECs produced VCAM1 and E-selectin protein in TEBVs with healthy or HGPS viSMCs. In summary, the viTEBV model has identified a role of the endothelium in HGPS.

Single-Cell RNA Sequencing Identifies Yes-Associated Protein 1-Dependent Hepatic Mesothelial Progenitors in Fibrolamellar Carcinoma.

Fibrolamellar carcinoma (FLC) is characterized by in-frame fusion of DnaJ heat shock protein family (Hsp40) member B1 (DNAJB1) with protein kinase cAMP-activated catalytic subunit α (PRKACA) and by dense desmoplasia. Surgery is the only effective treatment because mechanisms supporting tumor survival are unknown. We used single-cell RNA sequencing to characterize a patient-derived FLC xenograft model and identify therapeutic targets. Human FLC cells segregated into four discrete clusters that all expressed the oncogene Yes-associated protein 1 (YAP1). The two communities most enriched with cells coexpressing FLC markers [CD68, A-kinase anchoring protein 12 (AKAP12), cytokeratin 7, epithelial cell adhesion molecule (EPCAM), and carbamoyl palmitate synthase-1] also had the most cells expressing YAP1 and its proproliferative target genes (AREG and CCND1), suggesting these were proliferative FLC cell clusters. The other two clusters were enriched with cells expressing profibrotic YAP1 target genes, ACTA2, ELN, and COL1A1, indicating these were fibrogenic FLC cells. All clusters expressed the YAP1 target gene and mesothelial progenitor marker mesothelin, and many mesothelin-positive cells coexpressed albumin. Trajectory analysis predicted that the four FLC communities were derived from a single cell type transitioning among phenotypic states. After establishing a novel FLC cell line that harbored the DNAJB1-PRKACA fusion, YAP1 was inhibited, which significantly reduced expression of known YAP1 target genes as well as cell growth and migration. Thus, both FLC epithelial and stromal cells appear to arise from DNAJB1-PRKACA fusion in a YAP1-dependent liver mesothelial progenitor, identifying YAP1 as a target for FLC therapy.

Pharmacodynamic study of radium-223 in men with bone metastatic castration resistant prostate cancer.

BACKGROUND: Radium-223 is a targeted alpha-particle therapy that improves survival in men with metastatic castration resistant prostate cancer (mCRPC), particularly in men with elevated serum levels of bone alkaline phosphatase (B-ALP). We hypothesized that osteomimicry, a form of epithelial plasticity leading to an osteoblastic phenotype, may contribute to intralesional deposition of radium-223 and subsequent irradiation of the tumor microenvironment. METHODS: We conducted a pharmacodynamic study (NCT02204943) of radium-223 in men with bone mCRPC. Prior to and three and six months after radium-223 treatment initiation, we collected CTCs and metastatic biopsies for phenotypic characterization and CTC genomic analysis. The primary objective was to describe the impact of radium-223 on the prevalence of CTC B-ALP over time. We measured radium-223 decay products in tumor and surrounding normal bone during treatment. We validated genomic findings in a separate independent study of men with bone metastatic mCRPC (n = 45) and publicly accessible data of metastatic CRPC tissues. RESULTS: We enrolled 20 men with symptomatic bone predominant mCRPC and treated with radium-223. We observed greater radium-223 radioactivity levels in metastatic bone tumor containing biopsies compared with adjacent normal bone. We found evidence of persistent Cellsearch CTCs and B-ALP (+) CTCs in the majority of men over time during radium-223 therapy despite serum B-ALP normalization. We identified genomic gains in osteoblast mimicry genes including gains of ALPL, osteopontin, SPARC, OB-cadherin and loss of RUNX2, and validated genomic alterations or increased expression at the DNA and RNA level in an independent cohort of 45 men with bone-metastatic CRPC and in 150 metastatic biopsies from men with mCRPC. CONCLUSIONS: Osteomimicry may contribute in part to the uptake of radium-223 within bone metastases and may thereby enhance the therapeutic benefit of this bone targeting radiotherapy.

Engineering human pluripotent stem cells into a functional skeletal muscle tissue.

The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Here, we derive induced myogenic progenitor cells (iMPCs) via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. In 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells endogenously expressing Pax7. Under optimized 3D culture conditions, iMPCs derived from multiple hPSC lines reproducibly form functional skeletal muscle tissues (iSKM bundles) containing aligned multi-nucleated myotubes that exhibit positive force-frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. During 1-month culture, the iSKM bundles undergo increased structural and molecular maturation, hypertrophy, and force generation. When implanted into dorsal window chamber or hindlimb muscle in immunocompromised mice, the iSKM bundles survive, progressively vascularize, and maintain functionality. iSKM bundles hold promise as a microphysiological platform for human muscle disease modeling and drug development.

Research Resource: Roles for Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) in Systems Metabolism.

A number of epidemiological studies have implicated calcium (Ca(2+)) signaling as a major factor in obesity that contributes to aberrant systems metabolism. Somewhat paradoxically, obesity correlates with decreased circulating Ca(2+) levels, leading to increased release of intracellular Ca(2+) stores from the endoplasmic reticulum. These findings suggest that insulin resistance associated with the obese state is linked to activation of canonical Ca(2+) signaling pathways. Mechanistically, increased intracellular Ca(2+) binds calmodulin (CaM) to activate a set of Ca(2+)/CaM-dependent protein kinases. In this research resource, we explore the metabolic functions and implications of Ca(2+)/CaM-dependent protein kinase kinase 2 (CaMKK2) as a metabolic effector of Ca(2+)/CaM action. We reveal the importance of CaMKK2 for gating insulin release from pancreatic ß-cells while concomitantly influencing the sensitivity of insulin-responsive tissues. To provide a better understanding of the metabolic impact of CaMKK2 loss, we performed targeted metabolomic analyses of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice null for CaMKK2. We quantified amino acids and acyl carnitines in 3 insulin-sensitive tissues (liver, skeletal muscle, plasma) isolated from CaMKK2(-/-) mice and their wild-type littermates under conditions of dietary stress (low-fat diet, normal chow, high-fat diet, and fasting), thereby unveiling unique metabolic functions of CaMKK2. Our findings highlight CaMKK2 as a molecular rheostat for insulin action and emphasize the importance of Ca(2+)/CaM/CaMKK2 in regulation of whole-body metabolism. These findings reveal that CaMKK2 may be an attractive therapeutic target for combatting comorbidities associated with perturbed insulin signaling.

Pregnancy and Smoothelin-like Protein 1 (SMTNL1) Deletion Promote the Switching of Skeletal Muscle to a Glycolytic Phenotype in Human and Mice.

Pregnancy promotes physiological adaptations throughout the body, mediated by the female sex hormones progesterone and estrogen. Changes in the metabolic properties of skeletal muscle enable the female body to cope with the physiological challenges of pregnancy and may also be linked to the development of insulin resistance. We conducted global microarray, proteomic, and metabolic analyses to study the role of the progesterone receptor and its transcriptional regulator, smoothelin-like protein 1 (SMTNL1) in the adaptation of skeletal muscle to pregnancy. We demonstrate that pregnancy promotes fiber-type changes from an oxidative to glycolytic isoform in skeletal muscle. This phenomenon is regulated through an interaction between SMTNL1 and progesterone receptor, which alters the expression of contractile and metabolic proteins. smtnl1(-/-) mice are metabolically less efficient and show impaired glucose tolerance. Pregnancy antagonizes these effects by inducing metabolic activity and increasing glucose tolerance. Our results suggest that SMTNL1 has a role in mediating the actions of steroid hormones to promote fiber switching in skeletal muscle during pregnancy. Our findings also bear on the management of gestational diabetes that develops as a complication of pregnancy in ~4% of women.

Inhibition of calcium/calmodulin-dependent protein kinase kinase ß and calcium/calmodulin-dependent protein kinase IV is detrimental in cerebral ischemia.

BACKGROUND AND PURPOSE: Elevation of intracellular calcium was traditionally thought to be detrimental in stroke pathology. However, clinical trials testing treatments that block calcium signaling have failed to improve outcomes in ischemic stroke. Emerging data suggest that calcium may also trigger endogenous protective pathways after stroke. Calcium/calmodulin-dependent protein kinase kinase (CaMKK) is a major kinase activated by rising intracellular calcium. Compelling evidence has suggested that CaMKK and its downstream kinase CaMK IV are critical in neuronal survival when cells are under ischemic stress. We examined the functional role of CaMKK/CaMK IV signaling in stroke. METHODS: We used a middle cerebral artery occlusion model in mice. RESULTS: Our data demonstrated that pharmacological and genetic inhibition of CaMKK aggravated stroke injury. Additionally, deletion of CaMKK ß, one of the 2 CaMKK isoforms, reduced CaMK IV activation, and CaMK IV deletion in mice worsened stroke outcome. Finally, CaMKK ß or CaMK IV knockout mice had exacerbated blood-brain barrier disruption evidenced by increased hemorrhagic transformation and activation of matrix metalloproteinase. We observed transcriptional inactivation including reduced levels of histone deacetylase 4 phosphorylation in mice with CaMKK ß or CaMK IV deletion after stroke. CONCLUSIONS: Our data have established that the CaMKK/CaMK IV pathway is a key endogenous protective mechanism in ischemia. Our results suggest that this pathway serves as an important regulator of blood-brain barrier integrity and transcriptional activation of neuroprotective molecules in stroke.

A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53.

In the intrinsic pathway of apoptosis, cell-damaging signals promote the release of cytochrome c from mitochondria, triggering activation of the Apaf-1 and caspase-9 apoptosome. The ubiquitin E3 ligase MDM2 decreases the stability of the proapoptotic factor p53. We show that it also coordinated apoptotic events in a p53-independent manner by ubiquitylating the apoptosome activator CAS and the ubiquitin E3 ligase HUWE1. HUWE1 ubiquitylates the antiapoptotic factor Mcl-1, and we found that HUWE1 also ubiquitylated PP5 (protein phosphatase 5), which indirectly inhibited apoptosome activation. Breast cancers that are positive for the tyrosine receptor kinase HER2 (human epidermal growth factor receptor 2) tend to be highly aggressive. In HER2-positive breast cancer cells treated with the HER2 tyrosine kinase inhibitor lapatinib, MDM2 was degraded and HUWE1 was stabilized. In contrast, in breast cancer cells that acquired resistance to lapatinib, the abundance of MDM2 was not decreased and HUWE1 was degraded, which inhibited apoptosis, regardless of p53 status. MDM2 inhibition overcame lapatinib resistance in cells with either wild-type or mutant p53 and in xenograft models. These findings demonstrate broader, p53-independent roles for MDM2 and HUWE1 in apoptosis and specifically suggest the potential for therapy directed against MDM2 to overcome lapatinib resistance.

Deletion of CaMKK2 from the liver lowers blood glucose and improves whole-body glucose tolerance in the mouse.

Ca(2+)/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca(2+)/CaM-dependent protein kinase family that is expressed abundantly in brain. Previous work has revealed that CaMKK2 knockout (CaMKK2 KO) mice eat less due to a central nervous system -signaling defect and are protected from diet-induced obesity, glucose intolerance, and insulin resistance. However, here we show that pair feeding of wild-type mice to match food consumption of CAMKK2 mice slows weight gain but fails to protect from diet-induced glucose intolerance, suggesting that other alterations in CaMKK2 KO mice are responsible for their improved glucose metabolism. CaMKK2 is shown to be expressed in liver and acute, specific reduction of the kinase in the liver of high-fat diet-fed CaMKK2(floxed) mice results in lowered blood glucose and improved glucose tolerance. Primary hepatocytes isolated from CaMKK2 KO mice produce less glucose and have decreased mRNA encoding peroxisome proliferator-activated receptor γ coactivator 1-α and the gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, and these mRNA fail to respond specifically to the stimulatory effect of catecholamine in a cell-autonomous manner. The mechanism responsible for suppressed gene induction in CaMKK2 KO hepatocytes may involve diminished phosphorylation of histone deacetylase 5, an event necessary in some contexts for derepression of the peroxisome proliferator-activated receptor γ coactivator 1-α promoter. Hepatocytes from CaMKK2 KO mice also show increased rates of de novo lipogenesis and fat oxidation. The changes in fat metabolism observed correlate with steatotic liver and altered acyl carnitine metabolomic profiles in CaMKK2 KO mice. Collectively, these results are consistent with suppressed catecholamine-induced induction of gluconeogenic gene expression in CaMKK2 KO mice that leads to improved whole-body glucose homeostasis despite the presence of increased hepatic fat content.

The Ca2+/calmodulin-dependent protein kinase kinase, CaMKK2, inhibits preadipocyte differentiation.

When fed a standard chow diet, CaMKK2 null mice have increased adiposity and larger adipocytes than do wild-type mice, whereas energy balance is unchanged. Here, we show that Ca(2+)/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is expressed in preadipocytes, where it functions as an AMP-activated protein kinase (AMPK)α kinase. Acute inhibition or deletion of CaMKK2 in preadipocytes enhances their differentiation into mature adipocytes, which can be reversed by 5-aminoimidazole-4-carboxamide ribonucleotide-mediated activation of AMPK. During adipogenesis, CaMKK2 expression is markedly decreased and temporally accompanied by increases in mRNA encoding the early adipogenic genes CCAAT/enhancer binding protein (C/EBP) ß and C/EBP δ. Preadipocyte factor 1 has been reported to inhibit adipogenesis by up-regulating sex determining region Y-box 9 (Sox9) expression in preadipocytes and Sox9 suppresses C/EBPß and C/EBPδ transcription. We show that inhibition of the CaMKK2/AMPK signaling cascade in preadipocytes reduces preadipocyte factor 1 and Sox9 mRNA resulting in accelerated adipogenesis. We conclude that CaMKK2 and AMPK function in a signaling pathway that participates in the regulation of adiposity.

Gfer inhibits Jab1-mediated degradation of p27kip1 to restrict proliferation of hematopoietic stem cells.

Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27(kip1). In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27(kip1). KD of Gfer results in enhanced binding of p27(kip1) to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27(kip1) interaction. Furthermore, normalization of p27(kip1) in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27(kip1) pathway in HSCs that functions to restrict abnormal proliferation.

BDNF-mediated cerebellar granule cell development is impaired in mice null for CaMKK2 or CaMKIV.

The Ca(2+)/calmodulin-activated kinases CaMKK2 and CaMKIV are highly expressed in the brain where they play important roles in activating intracellular responses to elevated Ca(2+). To address the biological functions of Ca(2+) signaling via these kinases during brain development, we have examined cerebellar development in mice null for CaMKK2 or CaMKIV. Here, we demonstrate that CaMKK2/CaMKIV-dependent phosphorylation of cAMP response element-binding protein (CREB) correlates with Bdnf transcription, which is required for normal development of cerebellar granule cell neurons. We show in vivo and in vitro that the absence of either CaMKK2 or CaMKIV disrupts the ability of developing cerebellar granule cells in the external granule cell layer to cease proliferation and begin migration to the internal granule cell layer. Furthermore, loss of CaMKK2 or CaMKIV results in decreased CREB phosphorylation (pCREB), Bdnf exon I and IV-containing mRNAs, and brain-derived neurotrophic factor (BDNF) protein in cerebellar granule cell neurons. Reexpression of CaMKK2 or CaMKIV in granule cells that lack CaMKK2 or CaMKIV, respectively, restores pCREB and BDNF to wild-type levels and addition of BDNF rescues granule cell migration in vitro. These results reveal a previously undefined role for a CaMKK2/CaMKIV cascade involved in cerebellar granule cell development and show specifically that Ca(2+)-dependent regulation of BDNF through CaMKK2/CaMKIV is required for this process.

Calcium/calmodulin-dependent protein kinase (CaMK) IV mediates nucleocytoplasmic shuttling and release of HMGB1 during lipopolysaccharide stimulation of macrophages.

The chromatin-binding factor high-mobility group box 1 (HMGB1) functions as a proinflammatory cytokine and late mediator of mortality in murine endotoxemia. Although serine phosphorylation of HMGB1 is necessary for nucleocytoplasmic shuttling before its cellular release, the protein kinases involved have not been identified. To investigate if calcium/calmodulin-dependent protein kinase (CaMK) IV serine phosphorylates and mediates the release of HMGB1 from macrophages (Mphi) stimulated with LPS, RAW 264.7 cells or murine primary peritoneal Mphi were incubated with either STO609 (a CaMKIV kinase inhibitor), KN93 (a CaMKIV inhibitor), or we utilized cells from which CaMKIV was depleted by RNA interference (RNAi) before stimulation with LPS. We also compared the LPS response of primary Mphi isolated from CaMKIV(+/+) and CaMKIV(-/-) mice. In both cell types LPS induced activation and nuclear translocation of CaMKIV, which preceded HMGB1 nucleocytoplasmic shuttling. However, Mphi treated with KN93, STO609, or CaMKIV RNAi before LPS showed reduced nucleocytoplasmic shuttling of HMGB1 and release of HMGB1 into the supernatant. Additionally, LPS induced serine phosphorylation of HMGB1, which correlated with an interaction between CaMKIV and HMGB1 and with CaMKIV phosphorylation of HMGB1 in vitro. In cells, both HMGB1 phosphorylation and interaction with CaMKIV were inhibited by STO609 or CaMKIV RNAi. Similarly, whereas CaMKIV(+/+) Mphi showed serine phosphorylation of HMGB1 in response to LPS, this phosphorylation was attenuated in CaMKIV(-/-) Mphi. Collectively, our results demonstrate that CaMKIV promotes the nucleocytoplasmic shuttling of HMGB1 and suggest that the process may be mediated through CaMKIV-dependent serine phosphorylation of HMGB1.

Hypothalamic CaMKK2 contributes to the regulation of energy balance.

Detailed knowledge of the pathways by which ghrelin and leptin signal to AMPK in hypothalamic neurons and lead to regulation of appetite and glucose homeostasis is central to the development of effective means to combat obesity. Here we identify CaMKK2 as a component of one of these pathways, show that it regulates hypothalamic production of the orexigenic hormone NPY, provide evidence that it functions as an AMPKalpha kinase in the hypothalamus, and demonstrate that it forms a unique signaling complex with AMPKalpha and beta. Acute pharmacologic inhibition of CaMKK2 in wild-type mice, but not CaMKK2 null mice, inhibits appetite and promotes weight loss consistent with decreased NPY and AgRP mRNAs. Moreover, the loss of CaMKK2 protects mice from high-fat diet-induced obesity, insulin resistance, and glucose intolerance. These data underscore the potential of targeting CaMKK2 as a therapeutic intervention.

Calmodulin-dependent kinase IV links Toll-like receptor 4 signaling with survival pathway of activated dendritic cells.

Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin-dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4-/- DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.

Calmodulin-dependent protein kinase IV regulates hematopoietic stem cell maintenance.

The hematopoietic stem cell (HSC) gives rise to all mature, terminally differentiated cells of the blood. Here we show that calmodulin-dependent protein kinase IV (CaMKIV) is present in c-Kit+ ScaI+ Lin(-/low) hematopoietic progenitor cells (KLS cells) and that its absence results in hematopoietic failure, characterized by a diminished KLS cell population and by an inability of these cells to reconstitute blood cells upon serial transplantation. KLS cell failure in the absence of CaMKIV is correlated with increased apoptosis and proliferation of these cells in vivo and in vitro. In turn, these cell biological defects are correlated with decreases in CREB-serine 133 phosphorylation as well as in CREB-binding protein (CBP) and Bcl-2 levels. Re-expression of CaMKIV in Camk4-/- KLS cells results in the rescue of the proliferation defects in vitro as well as in the restoration of CBP and Bcl-2 to wild type levels. These studies show that CaMKIV is a regulator of HSC homeostasis and suggest that its effects may be in part mediated via regulation of CBP and Bcl-2.

Skeletal muscle adaptation in response to voluntary running in Ca2+/calmodulin-dependent protein kinase IV-deficient mice.

Mammalian skeletal muscles undergo adaptation in response to alteration in functional demands by means of a variety of cellular signaling events. Previous experiments in transgenic mice showed that an active form of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is capable of stimulating peroxisome proliferator-activated receptor gamma-coactivator 1alpha (PGC-1alpha) gene expression, promoting fast-to-slow fiber type switching and augmenting mitochondrial biogenesis in skeletal muscle. However, a role for endogenous CaMKIV in skeletal muscle has not been investigated rigorously. We report that genetically modified mice devoid of CaMKIV have normal fiber type composition and mitochondrial enzyme expression in fast-twitch skeletal muscles and responded to long-term (4 wk) voluntary running with increased expression of myosin heavy chain type IIa, myoglobin, PGC-1alpha, and cytochrome c oxidase IV proteins in plantaris muscle in a manner similar to that of wild-type mice. Short-term motor nerve stimulation (2 h at 10 Hz) likewise increased PGC-1alpha mRNA expression in tibialis anterior muscles in both Camk4(-/-) and wild-type mice. In addition, we have confirmed that no detectable CaMKIV protein is expressed in murine skeletal muscle. Thus CaMKIV is not required for the maintenance of slow-twitch muscle phenotype and endurance training-induced mitochondrial biogenesis and IIb-to-IIa fiber type switching in murine skeletal muscle. Other protein kinases sharing substrates with constitutively active CaMKIV may function as endogenous mediators of activity-dependent changes in myofiber phenotype.

Catalytic activity is required for calcium/calmodulin-dependent protein kinase IV to enter the nucleus.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a nuclear protein kinase that responds to acute rises in intracellular calcium by phosphorylating and activating proteins involved in transcription. Consistent with these roles, CaMKIV is found predominantly in the nucleus of cells in which it is expressed. Here we evaluate nuclear entry of CaMKIV and demonstrate that the protein kinase homology domain is both necessary and sufficient for nuclear localization. Unexpectedly, although catalytic activity is required for nuclear translocation, it is not required for CaMKIV to interact with the nuclear adaptor protein, importin-alpha. Because the catalytically inactive molecules remain in the cytoplasm, these data suggest that this interaction is not sufficient for nuclear entry. We evaluated a role for other proteins known to interact with CaMKIV in regulation of its nuclear entry. Although our data do not support a role for calmodulin or protein phosphatase 2A, the catalytically inactive CaMKIV proteins interact more avidly with CaM-dependent protein kinase kinase (CaMKK), which is restricted to the cytoplasm. We find that the catalytically inactive proteins do not inhibit nuclear entry of wild-type CaMKIV but do inhibit the ability of the wild-type protein kinase to stimulate cyclic AMP response element-binding protein-mediated transcription. Because activation loop phosphorylation is required for the transcriptional roles of CaMKIV, these data suggest that CaMKK phosphorylation of CaMKIV may occur in the cytoplasm. We propose that sequestration of CaMKK may be the molecular mechanism by which catalytically inactive mutants of CaMKIV exert their "dominant-negative" functions within the cell.

Calmodulin overexpression causes Ca(2+)-dependent apoptosis of pancreatic beta cells, which can be prevented by inhibition of nitric oxide synthase.

We investigated the mechanism of beta-cell loss in transgenic mice with elevated levels of beta cell calmodulin. The transgenic mice experienced a sudden rise in blood glucose levels between 21 and 28 days of age. This change was associated with development of severe hypoinsulinemia and loss of beta cells from the islets. Ultrastructural analysis revealed that compromised granule formation and apoptotic changes in the transgenic beta cells preceded the onset of hyperglycemia. Intraperitoneal injection of tolbutamide, an antidiabetic sulfonylurea, decreased blood glucose levels but increased the number of apoptotic beta cells. Finally, injection of transgenic mice with N(omega)-nitro-L-arginine methyl ester, which inhibits nitric oxide synthase activity, prevented hyperglycemia and lessened the changes in number and size of beta cells. Because immunofluorescent staining revealed preferential distribution of neural nitric oxide synthase in pancreatic beta cells, we speculate that overexpression of calmodulin sensitizes the beta cells to Ca(2+)-dependent activation of neural nitric oxide synthase, which mediates apoptosis.

Spectrophotometric analysis of solubilized rat hair proteins following intraperitoneal injection of 2,5-hexanedione.

The neurotoxic industrial solvents n-hexane and methyl n-butyl ketone are toxic by virtue of their common metabolite, 2,5-hexanedione (2,5-HD). Our previous work showed that pyrrole-like substances in solubilized rat hair proteins from rats injected (ip) daily with 2,5-HD demonstrated maximal absorbance in the 530-nm spectral region following reaction with Ehrlich's reagent (p-dimethylaminobenzaldehyde). Modification of the current analytical methods of achieving high specificity and lower detection limits with small sample quantities could have important implications for monitoring human populations. Adult male Sprague-Dawley rats were housed in individual metabolic cages with food and water provided ad libitum. Individual rats were injected (ip) daily with either 50 mg/kg 2,5-HD or physiologic-buffered saline (PBS). Plucked hair samples (dorsal, right flank, and left flank) were obtained from each rat before and at 7-day intervals after exposure to 2,5-HD or PBS for 28 days. Hair proteins solubilization and extraction procedures were adapted from earlier studies. We read 1 mL of dialyzed hair protein solution (2,5-HD or PBS control) against a reference cuvette containing water. Analyses utilized a Shimadzu UV 160 V recording spectrophotometer at an absorbency spectral range of 450 to 600 nm. In all spectral tracings, absorbance maxima (at 530 nm) characteristic of pyrrole-like substances were detected only in samples from 2,5-HD-treated rats. Absorbance at 530 nm was detected starting at Day 7 after exposure. The authors acknowledge Dr. Richard Whorton and Dr. Barbara Buckley for advice and for the use of their spectrophotometric equipment and Dr. Lowell A. Goldsmith for his help in our choice of the subject studied. This work was supported by the Walter P. Inman Memorial Fund in an award from Duke University to Dr. Leon Lack.