Binding and phosphorylation of a novel male germ cell-specific cGMP-dependent protein kinase-anchoring protein by cGMP-dependent protein kinase Ialpha

cGMP-dependent protein kinase (cGK) is a major cellular receptor of cGMP and plays important roles in cGMP-dependent signal transduction pathways. To isolate the components of the cGMP/cGK signaling pathway such as substrates and regulatory proteins of cGK, we employed the yeast two-hybrid system using cGK-Ialpha as a bait and isolated a novel male germ cell-specific 42-kDa protein, GKAP42 (42-kDa cGMP-dependent protein kinase anchoring protein). Although the N-terminal region (amino acids 1-66) of cGK-Ialpha is sufficient for the association with GKAP42, GKAP42 could not interact with cGK-Ibeta, cGK-II, or cAMP-dependent protein kinase. GKAP42 mRNA is specifically expressed in testis, where it is restricted to the spermatocytes and early round spermatids. Endogenous cGK-I is co-immunoprecipitated with anti-GKAP42 antibody from mouse testis tissue, suggesting that cGK-I physiologically interacts with GKAP42. Immunocytochemical observations revealed that GKAP42 is localized to the Golgi complex and that cGK-Ialpha is co-localized to the Golgi complex when coexpressed with GKAP42. Although both cGK-Ialpha and -Ibeta, but not cAMP-dependent protein kinase, phosphorylated GKAP42 in vitro, GKAP42 was a good substrate only for cGK-Ialpha in intact cells, suggesting that the association with kinase protein is required for the phosphorylation in vivo. Finally, we demonstrated that the kinase-deficient mutant of cGK-Ialpha stably associates with GKAP42 and that binding of cGMP to cGK-Ialpha facilitates their release from GKAP42. These findings suggest that GKAP42 functions as an anchoring protein for cGK-Ialpha and that cGK-Ialpha may participate in germ cell development through phosphorylation of Golgi-associated proteins such as GKAP42.     

Cyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type‐Iα (PKG‐Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG‐Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti‐apoptotic Bcl‐2‐related proteins in NCI‐H460 and A549 non‐small cell lung cancer (NSCLC) cells. 1H‐(1,2,4)oxadiazolo(4,3‐a)quinoxalin‐1‐one (ODQ), which blocks endogenous NO‐induced activation of cGMP/PKG‐Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c‐IAP1, livin, and survivin. DT‐2 (inhibitor of PKG‐Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c‐IAP1, livin, and survivin expression. Gene knockdown of PKG‐Iα expression using small‐interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c‐IAP1, livin, survivin, and Mcl‐1 expression. Inhibition of PKG‐Iα kinase activity with DT‐2 dramatically enhanced pro‐apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT‐2 and cisplatin increased apoptosis compared with cisplatin or DT‐2 alone, showing a synergistic effect. The data suggest that the PKG‐Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c‐IAP1, livin, survivin, and Mcl‐1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin. J. Cell. Biochem. 113: 3587–3598, 2012. © 2012 Wiley Periodicals, Inc.     

Tumor Necrosis Factor (TNF)-α-induced Repression of GKAP42 Protein Levels through cGMP-dependent Kinase (cGK)-Iα Causes Insulin Resistance in 3T3-L1 Adipocytes

Insulin receptor substrates (IRSs) have been shown to be major mediators of insulin signaling. Recently, we found that IRSs form high-molecular weight complexes, and here, we identify by yeast two-hybrid screening a novel IRS-1-associated protein: a 42-kDa cGMP-dependent protein kinase-anchoring protein (GKAP42). GKAP42 knockdown in 3T3-L1 adipocytes suppressed insulin-dependent IRS-1 tyrosine phosphorylation and downstream signaling, resulting in suppression of GLUT4 translocation to plasma membrane induced by insulin. In addition, GLUT4 translocation was also suppressed in cells overexpressing GKAP42-N (the IRS-1 binding region of GKAP42), which competed with GKAP42 for IRS-1, indicating that GKAP42 binding to IRS-1 is required for insulin-induced GLUT4 translocation. Long term treatment of 3T3-L1 adipocytes with TNF-α, which induced insulin resistance, significantly decreased the GKAP42 protein level. We then investigated the roles of cGMP-dependent kinase (cGK)-Iα, which bound to GKAP42, in these changes. cGK-Iα knockdown partially rescued TNF-α-induced decrease in GKAP42 and impairment of insulin signals. These data indicated that TNF-α-induced repression of GKAP42 via cGK-Iα caused reduction of insulin-induced IRS-1 tyrosine phosphorylation at least in part. The present study describes analysis of the novel TNF-α-induced pathway, cGK-Iα-GKAP42, which regulates insulin-dependent signals and GLUT4 translocation.     

SPATC1L maintains the integrity of the sperm head‐tail junction

Spermatogenesis is a tightly regulated process involving germ cell‐specific and germ cell‐predominant genes. Here we investigate a novel germ cell‐specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9‐mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z‐line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L‐PKA complex in maintaining the stability of the sperm head‐tail junction, thereby revealing a new molecular basis for sperm head‐tail integrity.     

Functional regulation of transient receptor potential canonical 7 by cGMP-dependent protein kinase Iα

The cGMP/cGMP-dependent protein kinase (cGK) signaling pathway is implicated in the functional regulation of intracellular calcium levels. In the present study, we investigated the regulation of transient receptor potential canonical 7 (TRPC7) by the cGMP/cGK-I pathway. TRPC7 contains three putative cGK phosphorylation sites (Arg-Arg/Lys-Xaa-Ser/Thr). However, the role of cGK-I in the regulation of TRPC7 activity remains unclear. In vitro and in vivo kinase assays have revealed that cGK-Iα phosphorylates mouse TRPC7 but not mouse TRPC3. Site-directed mutagenesis analysis revealed that TRPC7 was phosphorylated by cGK-Iα at threonine 15. Phosphorylation of TRPC7 significantly suppressed carbachol-induced calcium influx and CREB phosphorylation. Furthermore, co-immunoprecipitation assay demonstrated that cGK-Iα interacted with the ankyrin repeat domain in the N terminus of TRPC7. cGK-Iβ also bound to TRPC7, while the type II regulatory subunit of cAMP-dependent protein kinase did not bind. These data indicate that cGK-Iα interacts with and phosphorylates TRPC7, contributing to the quick and accurate regulation of calcium influx and CREB phosphorylation.     

TopBP1 deficiency impairs the localization of proteins involved in early recombination and results in meiotic chromosome defects during spermatogenesis

Topoisomerase IIβ-binding protein 1 (TopBP1) is BRCT domain-containing protein that is required for DNA double-strand break (DSB) repair and DNA damage responses; however, its function during the early stage of spermatogenesis is still unclear. To investigate the physiological role of TopBP1, we have generated germ cell-specific TopBP1-depleted mouse model. TopBP1-deleted mice were infertile, showed a loss of germ cells and had meiotic defects. Conditional TopBP1 deletion resulted in reduced testis size, reduced number of epididymal sperm, increased apoptosis, and severely compromised fertility. TopBP1 deficiency caused defects in DMC1 and Rad51 foci formation, abnormal synaptonemal complexes and meiotic chromosome defects. Collectively, these results suggest that TopBP1 deficiency during spermatogenesis impairs the localization of proteins involved in early recombination at DSBs, results in meiotic chromosome defects and leads to infertility.     

Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression,Functionality and Involvement in Motility Regulation

Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility.     

Age-dependent loss of sperm production in mice via impaired lysophosphatidic acid signaling

Approximately half of all infertility cases can be attributed to male reproductive dysfunction for which low sperm count is a major contributing factor. The current study identified receptor-mediated lysophosphatidic acid (LPA) signaling as a new molecular component influencing male fertility. LPA is a small signaling phospholipid, the effects of which are mediated through at least five G protein-coupled receptors, named LPA 1-5. LPA1/2/3, but not LPA4/5, show high expression in mouse testis. Mice deficient in LPA1/2/3 showed a testosterone-independent reduction of mating activity and sperm production, with an increased prevalence of azoospermia in aging animals. A significant increase of germ cell apoptosis also was observed in testes. Germ cell apoptosis led to a reduction in germ cell proliferation. These data demonstrate a novel in vivo function for LPA signaling as a germ cell survival factor during spermatogenesis.     

Cyclic cytidine 3′,5′-monophosphate (cCMP) signals via cGMP kinase I

We analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane-permeable cCMP analogue dibutyryl-cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP-dependent protein kinase (cGK) Iα and Iβ and stimulated cGK in aorta lysates. cCMP-induced relaxation was abolished in cGKI-knockout tissue. Additionally, deletion of inositol-trisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP-mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP-mediated inhibition of platelet aggregation was absent in cGKI- and IRAG-deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.     

Glucagon‐like peptide‐1 ameliorates cardiac lipotoxicity in diabetic cardiomyopathy via the PPARα pathway

Lipotoxicity cardiomyopathy is the result of excessive accumulation and oxidation of toxic lipids in the heart. It is a major threat to patients with diabetes. Glucagon‐like peptide‐1 (GLP‐1) has aroused considerable interest as a novel therapeutic target for diabetes mellitus because it stimulates insulin secretion. Here, we investigated the effects and mechanisms of the GLP‐1 analog exendin‐4 and the dipeptidyl peptidase‐4 inhibitor saxagliptin on cardiac lipid metabolism in diabetic mice (DM). The increased myocardial lipid accumulation, oxidative stress, apoptosis, and cardiac remodeling and dysfunction induced in DM by low streptozotocin doses and high‐fat diets were significantly reversed by exendin‐4 and saxagliptin treatments for 8 weeks. We found that exendin‐4 inhibited abnormal activation of the (PPARα)‐CD36 pathway by stimulating protein kinase A (PKA) but suppressing the Rho‐associated protein kinase (ROCK) pathway in DM hearts, palmitic acid (PA)‐treated rat h9c2 cardiomyocytes (CMs), and isolated adult mouse CMs. Cardioprotection in DM mediated by exendin‐4 was abolished by combination therapy with the PPARα agonist wy‐14643 but mimicked by PPARα gene deficiency. Therefore, the PPARα pathway accounted for the effects of exendin‐4. This conclusion was confirmed in cardiac‐restricted overexpression of PPARα mediated by adeno‐associated virus serotype‐9 containing a cardiac troponin T promoter. Our results provide the first direct evidence that GLP‐1 protects cardiac function by inhibiting the ROCK/PPARα pathway, thereby ameliorating lipotoxicity in diabetic cardiomyopathy.     

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.     

Ehd4 is required to attain normal prepubertal testis size but dispensable for fertility in male mice

The four highly homologous members of the C‐terminal EH domain‐containing (EHD) protein family (EHD1‐4) regulate endocytic recycling. To delineate the role of EHD4 in normal physiology and development, mice with a conditional knockout of the Ehd4 gene were generated. PCR of genomic DNA and Western blotting of organ lysates from Ehd4^−/− mice confirmed EHD4 deletion. Ehd4^−/− mice were viable and born at expected Mendelian ratios; however, males showed a 50% reduction in testis weight, obvious from postnatal day 31. An early (Day 10) increase in germ cell proliferation and apoptosis and a later increase in apoptosis (Day 31) were seen in the Ehd4^−/− testis. Other defects included a progressive reduction in seminiferous tubule diameter, dysregulation of seminiferous epithelium, and head abnormalities in elongated spermatids. As a consequence, lower sperm counts and reduced fertility were observed in Ehd4^−/− males. Interestingly, EHD protein expression was seen to be temporally regulated in the testis and EHD4 levels peaked between days 10 and 15. In the adult testis, EHD4 was highly expressed in primary spermatocytes and EHD4 deletion altered the levels of other EHD proteins in an age‐dependent manner. We conclude that high levels of EHD1 in the adult Ehd4^−/− testis functionally compensate for lack of EHD4 and prevents the development of severe fertility defects. Our results suggest a role for EHD4 in the proper development of postmitotic and postmeiotic germ cells and implicate EHD protein‐mediated endocytic recycling as an important process in germ cell development and testis function. genesis 48:328–342, 2010. © 2010 Wiley‐Liss, Inc.     

Murine male germ cell apoptosis induced by busulfan treatment correlates with loss of c-kit-expression in a Fas/FasL- and p53-independent manner

Male germ cell apoptosis has been extensively explored in rodents. In contrast, very little is known about the susceptibility of developing germ cells to apoptosis in response to busulfan treatment. Spontaneous apoptosis of germ cells is rarely observed in the adult mouse testis, but under the experimental conditions described here, busulfan-treated mice exhibited a marked increase in apoptosis and a decrease in testis weight. TdT-mediated dUTP-X nicked end labeling analysis indicates that at one week following busulfan treatment, apoptosis was confined mainly to spermatogonia, with lesser effects on spermatocytes. The percentage of apoptosis-positive tubules and the apoptotic cell index increased in a time-dependent manner. An immediate effect was observed in spermatogonia within one week of treatment, and in the following week, secondary effects were observed in spermatocytes. RT-PCR analysis showed that expression of the spermatogonia-specific markers c-kit and Stra 8 was reduced but that Gli I gene expression remained constant, which is indicative of primary apoptosis of differentiating type A spermatogonia. Three and four weeks after busulfan treatment, RAD51 and FasL expression decreased to nearly undetectable levels, indicating that meiotic spermatocytes and post-meiotic cells, respectively, were lost. The period of germ cell depletion did not coincide with increased p53 or Fas/FasL expression in the busulfan-treated testis, although p110Rb phosphorylation and PCNA expression were inhibited. These data suggest that increased depletion of male germ cells in the busulfan-treated mouse is mediated by loss of c-kit/SCF signaling but not by p53- or Fas/FasL-dependent mechanisms. Spermatogonial stem cells may be protected from cell death by modulating cell cycle signaling such that E2F-dependent protein expression, which is critical for G1 phase progression, is inhibited.     

Elements of the nitric oxide/cGMP pathway expressed in cerebellar granule cells: biochemical and functional characterisation

It is known that the nitric oxide (NO)/cGMP pathway affects neuronal development and the expression of the different proteins is developmentally dependent in several brain areas. However, so far there are no data on the expression of the proteins involved in this signalling system during the development of the cerebellar granule cell, one of the most widely used models of neuronal development. This study was accordingly designed to analyse the developmental regulation of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase subunits (alpha1, alpha2 and beta1) and cGMP-dependent protein kinases (cGK I and cGK II) in cerebellar granule cells through real time-polymerase chain reaction (RT-PCR) and Western blotting. We were able to detect guanylyl cyclase subunits and cGK I and cGK II in cerebellar granule cells at every stage of development examined (cells freshly isolated from 7-day-old rat pups, and cells cultured for 7 days or 14 days). Expression levels, nevertheless, varied significantly at each stage. nNOS, alpha2 and beta1 and cGK II levels increased during granule cell development, while alpha1 and cGK I showed an opposite behaviour pattern; the levels of these latter proteins diminished as the cells matured. The functionality of this pathway was assessed by stimulating cells kept in culture for 7 days with DEA/NO or with N-methyl-D-aspartate (NMDA). Cells responded by increasing intracellular cGMP and activating cGMP-dependent protein kinase activity, which effectively phosphorylated two well-known substrates of this activity, the vasodilator stimulated phosphoprotein (VASP) and the cAMP response element binding protein (CREB). In summary, through both functional and biochemical tests, this is the first demonstration of a complete NO/cGMP signalling transduction pathway in cerebellar granule cells. Our results also indicate the developmental regulation of the proteins in this system.