Ultrastructure of Highly Ordered Granules in Alveolar Type II Cells in Several Species.

Alveolar Type II cells from seven mammalian species were examined for a protein in the rough endoplasmic reticulum (RER), which showed a multilayered, repeating motif. Each motif, 100 nm in width, comprised two parallel outer dense layers, a less dense central layer, and often 1-3 faint layers on either side of the latter. Outer layers showed periodicities at 3-4 densities/100 nm of width, while layers on either side of the central layer showed 5-7 densities/100 nm of width. RER membranes were ribosome-free when parallel to these layers, but showed four ribosomes per motif at the growing ends: one ribosome at each outer dense layer, and one on either side of the less dense central layer. Granules appeared as single or as multiple motifs, stacked, curved, folded, or branching together within the same RER profile. Hexagons of around 30 nm in diameter with central densities were seen in tangential cuts of outer dense layers. Granule incidence varied: guinea pig > ferret > dog. Possible homologous structures occurred in rabbit and cat, but not in rat or mouse. Surfactant protein A (SP-A), a C-type lectin produced in Type II cells, forms trimers and bouquet-like 18-mer and can oligomerize further. Two pairs of SP-A 18-mers with carbohydrate recognition domains pointing inwardly and outwardly, stacked vertically as a column of four molecules, then repeated side by side in rows, approximated the size and layering patterns observed in these granules. Sequence analyses of SP-A from these species showed phylogenetic distances consistent with the observed occurrence and frequency of patterned granules. Anat Rec, 301:1290-1302, 2018. © 2018 Wiley Periodicals, Inc.

Oral cadmium in mice carrying 5 versus 2 copies of the Slc39a8 gene: comparison of uptake, distribution, metal content, and toxicity.

The highly conserved human and mouse SLC39A8 gene encodes the divalent cation/bicarbonate symporter ZIP8 expressed ubiquitously in most cell types. Our bacterial artificial chromosome-transgenic BTZIP8-3 line has 3 additional copies of the Slc39a8 gene in addition to its constitutive diploid pair found in wild-type (WT) mice. In liver, kidney, lung, testis, gastrointestinal tract, and brain, BTZIP8-3 mice are known to express ∼2.5 times greater amounts of ZIP8, compared with WT mice. Herein we administered cadmium chloride (CdCl2) in drinking water (100 mg/L through week 2, 200 mg/L through week 4, 400 mg/L through week 8, 800 mg/L through week 12, and 1600 mg/L through week 20, when the experiment was concluded). We postulated that Cd uptake and distribution--and, therefore, toxicity in certain tissues--would be enhanced in BTZIP8-3, compared with WT mice. BTZIP8-3 and WT groups ingested comparable amounts of Cd. Compared with WT, BTZIP8-3 mice showed tissue specific: increases in Cd, zinc, and manganese content and decreases in calcium content. Both Cd-exposed BTZIP8-3 and WT were similar in lower urinary pH; increased plasma alanine and aspartate aminotransferase activities; elevated iron and copper content in liver, kidney, lung, and testis; and higher blood urea nitrogen and kidney weight. Histological changes in liver, kidney, lung, and testis were minimal. In summary, at the daily oral Cd exposures chosen for this study, 5 versus 2 Slc39a8 gene copies result in no differences in Cd toxicity but do cause differences in tissue-specific content of Cd, zinc, manganese, calcium, iron, and copper.

Loss of NHE1 activity leads to reduced oxidative stress in heart and mitigates high-fat diet-induced myocardial stress.

Acute inhibition of the NHE1 Na(+)/H(+) exchanger protects against ischemia-reperfusion injury and chronic inhibition attenuates development of cardiac hypertrophy and failure. To determine the cardiac effects of chronic inhibition of NHE1 under non-pathological conditions we used NHE1-null mice as a model of long-term NHE1 inhibition. Cardiovascular performance was relatively normal in Nhe1(-/-) mice although cardiac contractility and relaxation were slightly improved in mutant mice of the FVB/N background. GSH levels and GSH:GSSG ratios were elevated in Nhe1(-/-) hearts indicating an enhanced redox potential. Consistent with a reduced need for antioxidant protection, expression of heat shock proteins Hsp60 and Hsp25 was lower in Nhe1(-/-) hearts. Similarly, expression of mitochondrial superoxide dismutase 2 was reduced, with no increase in expression of other ROS scavenging enzymes. GLUT1 levels were increased in Nhe1(-/-) hearts, the number of lipid droplets in myocytes was reduced, and PDK4 expression was refractory to high-fat diet-induced upregulation observed in wild-type hearts. High-fat diet-induced stress was attenuated in Nhe1(-/-) hearts, as indicated by smaller increases in phosphorylation of Hsp25 and α-B crystallin, and there was better preservation of insulin sensitivity, as evidenced by PKB/Akt phosphorylation. Plasma glucose and insulin levels were lower and high-fat diet-induced hepatic lipid accumulation was reduced in Nhe1(-/-) mice, demonstrating extracardiac effects of NHE1 ablation. These data indicate that long-term ablation of NHE1 activity increases the redox potential, mitigates high-fat diet-induced myocardial stress and fatty liver disease, leads to better preservation of insulin sensitivity, and may alter both cardiac and systemic metabolic substrate handling in mice.

Oral benzo[a]pyrene in Cyp1a1/1b1(-/-) double-knockout mice: Microarray analysis during squamous cell carcinoma formation in preputial gland duct.

Benzo[a]pyrene (BaP) is a prototypical polycyclic aromatic hydrocarbon (PAH) found in combustion processes. Cytochrome P450 1A1 and 1B1 enzymes (CYP1A1, CYP1B1) and other enzymes can activate PAHs to reactive oxygenated intermediates involved in mutagenesis and tumor initiation; also, CYP1 enzymes can detoxify PAHs. Cyp1(+/+) wild-type (WT) and Cyp1b1(-/-) knockout mice receiving oral BaP (12.5 mg/kg/day) remain healthy for >12 months. In contrast, we found that global knockout of the Cyp1a1 gene (1a1KO) results in proximal small intestine (PSI) adenocarcinoma within 8-12 weeks on this BaP regimen; striking compensatory increases in PSI CYP1B1 likely participate in initiation of adenocarcinoma in 1a1KO mice. Cyp1a1/1b1(-/-) double-knockout (DKO) mice on this BaP regimen show no PSI adenocarcinoma, but instead preputial gland duct (PGD) squamous cell carcinoma (SCC) occurs by 12 weeks. Herein, we compare microarray expression of PGD genes in WT, 1a1KO and DKO mice at 0, 4, 8, 12 and 16 weeks of oral BaP; about four dozen genes up- or down-regulated during most critical time-points were further verified by qRT-PCR. In DKO mice, CYP3A59 was unequivocally identified as the BaP-inducible and BaP-metabolizing best candidate responsible for initiation of BaP-induced SCC. Striking increases or decreases were found in 26 cancer-related genes plus eight Serpin genes in DKO, but not in 1a1KO or WT, mice on this BaP regimen; of the 26, 8 were RAS-related oncogenes. The mechanism by which cancer-related genes are responsible for SCC tumor progression in the PGD remains to be elucidated.

ZIP8 zinc transporter: indispensable role for both multiple-organ organogenesis and hematopoiesis in utero.

Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn(2+)/(HCO(3)(-))(2) symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects-proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis.

Identification of a conserved anti-apoptotic protein that modulates the mitochondrial apoptosis pathway.

Here we identified an evolutionarily highly conserved and ubiquitously expressed protein (C9orf82) that shows structural similarities to the death effector domain of apoptosis-related proteins. RNAi knockdown of C9orf82 induced apoptosis in A-549 and MCF7/casp3-10b lung and breast carcinoma cells, respectively, but not in cells lacking caspase-3, caspase-10 or both. Apoptosis was associated with activated caspases-3, -8, -9 and -10, and inactivation of caspases 10 or 3 was sufficient to block apoptosis in this pathway. Apoptosis upon knockdown of C9orf82 was associated with increased caspase-10 expression and activation, which was required for the generation of an 11 kDa tBid fragment and activation of Caspase-9. These data suggest that C9orf82 functions as an anti-apoptotic protein that modulates a caspase-10 dependent mitochondrial caspase-3/9 feedback amplification loop. We designate this ubiquitously expressed and evolutionarily conserved anti-apoptotic protein Conserved Anti-Apoptotic Protein (CAAP). We also demonstrated that treatment of MCF7/casp3-10b cells with staurosporine and etoposides induced apoptosis and knockdown of CAAP expression. This implies that the CAAP protein could be a target for chemotherapeutic agents.

Secretory pathway stress responses as possible mechanisms of disease involving Golgi Ca2+ pump dysfunction.

In mammalian tissues, uptake of Ca(2+) and Mn(2+) by Golgi membranes is mediated by the secretory pathway Ca(2+) -ATPases, SPCA1 and SPCA2, encoded by the ATP2C1 and ATP2C2 genes. Loss of one copy of the ATP2C1 gene, which causes SPCA1 haploinsufficiency, leads to squamous cell tumors of keratinized epithelia in mice and to Hailey-Hailey disease, an acantholytic skin disease, in humans. Although the disease phenotypes resulting from SPCA1 haploinsufficiency in mice and humans are quite different, each species-specific phenotype is remarkably similar to those arising as a result of null mutations in one copy of the ATP2A2 gene, encoding SERCA2, the endoplasmic reticulum (ER) Ca(2+) pump. SERCA2 haploinsufficiency, like SPCA1 haploinsufficiency, causes squamous cell tumors in mice and Darier's disease, also an acantholytic skin disease, in humans. The phenotypic similarities between SPCA1 and SERCA2 haploinsufficiency in the two species, and the general functions of the two pumps in consecutive compartments of the secretory pathway, suggest that the underlying disease mechanisms are similar. In this review, we discuss evidence supporting the view that chronic Golgi stress and/or ER stress resulting from Ca(2+) pump haploinsufficiencies leads to activation of cellular stress responses in keratinocytes, with the predominance of proapoptotic pathways (although not necessarily apoptosis itself) leading to acantholytic skin disease in humans and the predominance of prosurvival pathways leading to tumors in mice.

Loss of the NHE2 Na+/H+ exchanger in mice results in dilation of folliculo-stellate cell canaliculi.

Genetic ablation of the NHE2 Na+/H+ exchanger causes gastric achlorhydria, absorptive defects in kidney and colon, and low fertility. Here we show that NHE2 is expressed in the pituitary, with the highest mRNA expression in pars distalis and lower expression in pars intermedia. In pars distalis of NHE2-null mice, prominent cyst-like dilatations of folliculo-stellate (FS) cell canaliculi developed with age, and there were increased FS cell area, accumulation of lipid in FS cell cytoplasm, redundancies in FS cell basement membrane, and other changes. The expansion of the canaliculi indicates that NHE2 is a major absorptive Na+/H+ exchanger in the luminal membranes lining the extensive network of channels formed by FS cells, which may provide a means of intrapituitary communication. The results suggest that NHE2 contributes to homeostatic regulation of the volume and composition of the canalicular fluid and may counter the secretory activity of the CFTR Cl⁻ channel, which is known to be expressed in pituitary.

Loss of the AE3 anion exchanger in a hypertrophic cardiomyopathy model causes rapid decompensation and heart failure.

The AE3 Cl(-)/HCO(3)(-) exchanger is abundantly expressed in the sarcolemma of cardiomyocytes, where it mediates Cl(-)-uptake and HCO(3)(-)-extrusion. Inhibition of AE3-mediated Cl(-)/HCO(3)(-) exchange has been suggested to protect against cardiac hypertrophy; however, other studies indicate that AE3 might be necessary for optimal cardiac function. To test these hypotheses we crossed AE3-null mice, which appear phenotypically normal, with a hypertrophic cardiomyopathy mouse model carrying a Glu180Gly mutation in α-tropomyosin (TM180). Loss of AE3 had no effect on hypertrophy; however, survival of TM180/AE3 double mutants was sharply reduced compared with TM180 single mutants. Analysis of cardiac performance revealed impaired cardiac function in TM180 and TM180/AE3 mutants. TM180/AE3 double mutants were more severely affected and exhibited little response to ß-adrenergic stimulation, a likely consequence of their more rapid progression to heart failure. Increased expression of calmodulin-dependent kinase II and protein phosphatase 1 and differences in methylation and localization of protein phosphatase 2A were observed, but were similar in single and double mutants. Phosphorylation of phospholamban on Ser16 was sharply increased in both single and double mutants relative to wild-type hearts under basal conditions, leading to reduced reserve capacity for ß-adrenergic stimulation of phospholamban phosphorylation. Imaging analysis of isolated myocytes revealed reductions in amplitude and decay of Ca(2+) transients in both mutants, with greater reductions in TM180/AE3 mutants, consistent with the greater severity of their heart failure phenotype. Thus, in the TM180 cardiomyopathy model, loss of AE3 had no apparent anti-hypertrophic effect and led to more rapid decompensation and heart failure.

In utero and lactational exposure to a complex mixture of polychlorinated biphenyls: toxicity in pups dependent on the Cyp1a2 and Ahr genotypes.

Polychlorinated biphenyls (PCBs) are persistent toxic pollutants occurring as complex mixtures in the environment. Humans are known genetically to have > 60-fold differences in hepatic cytochrome P450 1A2 (CYP1A2) levels and > 12-fold differences in aryl hydrocarbon receptor (AHR) affinity, both of which could affect PCB pharmacokinetics. Thus, we compared Ahr(b1)_Cyp1a2(+/+) high-affinity AHR wild-type, Ahr(d)_Cyp1a2(+/+) poor affinity AHR wild-type, Ahr(b1)_Cyp1a2(-/-) knockout, and Ahr(d)_Cyp1a2(-/-) knockout mouse lines. We chose a mixture of three coplanar and five noncoplanar PCBs to reproduce that seen in human tissues, breast milk, and the food supply. The mixture was given by gavage to the mother on gestational day 10.5 (GD10.5) and postnatal day 5 (PND5); tissues were collected from pups and mothers at GD11.5, GD18.5, PND6, PND13, and PND28. Ahr(b1)_Cyp1a2(-/-) pups showed lower weight at birth and slower rate of growth postnatally. Absence of CYP1A2 resulted in significant splenic atrophy at PND13 and PND28. Presence of high-affinity AHR enhanced thymic atrophy and liver hypertrophy in the pups. Concentrations of each congener were analyzed at all time points: maximal noncoplanar congener levels in maternal tissues were observed from GD18 until PND6, whereas the highest levels in pups were found between PND6 and PND28. Coplanar PCB concentrations were generally higher in Ahr(d)-containing pup tissues; these findings are consistent with earlier studies demonstrating the crucial importance of AHR-mediated inducible CYP1 in the gastrointestinal tract as a means of detoxication of oral planar polycyclic aromatic hydrocarbons.