scholarly journals Effects of Carboxypeptidase E Overexpression on Insulin mRNA Levels, Regulated Insulin Secretion, and Proinsulin Processing of Pituitary GH3 Cells Transfected with a Furin-Cleavable Human Proinsulin cDNA

2002 ◽  
Vol 11 (8) ◽  
pp. 803-811 ◽  
Author(s):  
Luca Polastri ◽  
Francesca Galbiati ◽  
Franco Folli ◽  
Alberto M. Davalli
Keyword(s):  
Insulin Secretion ◽  
Cell Lines ◽  
Secretory Granules ◽  
Gh3 Cells ◽  
Soluble Form ◽  
Mrna Levels ◽  
Carboxypeptidase E ◽  
Proinsulin Processing ◽  
Membrane Bound ◽  
Overexpressing Cell

We recently developed two rat pituitary GH3 cell clones engineered to secrete human insulin (InsGH3). InsGH3 cells convert proinsulin into mature insulin, which is partially stored into a readily releasable pool of secretory granules. The efficiency of these processes, however, is relatively low in these cells, either in vitro or in vivo. This study was aimed at determining whether carboxypeptidase E (Cpe) overexpression can increase proinsulin processing and regulated secretion by InsGH3 clones. Indeed, in its membrane-bound form Cpe works as sorting receptor for the regulated secretory pathway of many hormones while, in its soluble form, Cpe takes part to the late step of insulin maturation. We obtained two Cpe-overexpressing cell lines from two different InsGH3 clones (InsGH3/C1 and C7). In the Cpe-overexpressing cell lines, derived from InsGH3 of clone 1 (InsGH3/C1-HACpe), in which the membrane-bound form of exogenous Cpe is accounted for by 90% of total Cpe immunoreactivity, we observed an increase in proinsulin gene expression, and in basal and stimulated insulin secretion compared with the original clone. In contrast, in the Cpe-overexpressing cell line derived from InsGH3 of clone 7 (InsGH3/C7-HACpe), where the exogenous membrane-bound form was only 60% of total Cpe, we detected a decrease in basal insulin release and a modest, albeit significant, increase in intracellular proinsulin processing. In conclusion, Cpe overexpression can increase regulated insulin secretion and proinsulin processing in InsGH3 cells; however, such improvements appear quantitatively and qualitatively modest.

scholarly journals Inside the Insulin Secretory Granule

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 515
Author(s):  
Mark Germanos ◽  
Andy Gao ◽  
Matthew Taper ◽  
Belinda Yau ◽  
Melkam A. Kebede
Keyword(s):  
Secretory Granules ◽  
Β Cells ◽  
Β Cell ◽  
Proinsulin Processing ◽  
Membrane Bound ◽  
Golgi Network ◽  
Insulin Secretory Granule ◽  
Insulin Storage ◽  
Cellular Stimulation

The pancreatic β-cell is purpose-built for the production and secretion of insulin, the only hormone that can remove glucose from the bloodstream. Insulin is kept inside miniature membrane-bound storage compartments known as secretory granules (SGs), and these specialized organelles can readily fuse with the plasma membrane upon cellular stimulation to release insulin. Insulin is synthesized in the endoplasmic reticulum (ER) as a biologically inactive precursor, proinsulin, along with several other proteins that will also become members of the insulin SG. Their coordinated synthesis enables synchronized transit through the ER and Golgi apparatus for congregation at the trans-Golgi network, the initiating site of SG biogenesis. Here, proinsulin and its constituents enter the SG where conditions are optimized for proinsulin processing into insulin and subsequent insulin storage. A healthy β-cell is continually generating SGs to supply insulin in vast excess to what is secreted. Conversely, in type 2 diabetes (T2D), the inability of failing β-cells to secrete may be due to the limited biosynthesis of new insulin. Factors that drive the formation and maturation of SGs and thus the production of insulin are therefore critical for systemic glucose control. Here, we detail the formative hours of the insulin SG from the luminal perspective. We do this by mapping the journey of individual members of the SG as they contribute to its genesis.

Calcium channels in excitable cells: divergent genotypic and phenotypic expression of alpha 1-subunits

1994 ◽  
Vol 267 (2) ◽  
pp. C411-C424 ◽  
Author(s):  
A. Lievano ◽  
A. Bolden ◽  
R. Horn
Keyword(s):  
Rat Brain ◽  
Cell Lines ◽  
Phenotypic Expression ◽  
Gh3 Cells ◽  
Mrna Levels ◽  
Excitable Cells ◽  
Patch Clamp Recording ◽  
Whole Cell Patch Clamp ◽  
Ribonuclease Protection ◽  
P Type

The Ba2+ currents and mRNA levels of four members of the rat brain family of alpha 1-subunit Ca2+ channel genes were examined and compared in the rat cell lines GH3 and PC-12 and in the mouse lines NIE-115 and AtT-20. The RNA was measured with ribonuclease protection assays using probes derived from rat brain (rb) Ca2+ channel cDNAs (rbA, rbB, rbC, and rbD), and the Ba2+ currents were studied by whole cell patch-clamp recording. L-, N-, P-, and T-type currents were discriminated by the voltage dependence and pharmacological properties of Ba2+ currents. All cell lines expressed all four rat brain Ca2+ channel genes, except GH3 cells, which lacked rbB. The functional diversity of Ba2+ currents, however, was quite different among the cell lines. GH3 cells showed evidence of L- and T-type currents, undifferentiated PC-12 cells of L-type currents, AtT-20 cells of L-, N-, and P-type currents, and undifferentiated NIE-115 cells of a T-type current that was partially blocked by both nifedipine and BAY K 8644. Dimethyl sulfoxide-differentiated NIE-115 cells also had an L-type current. Differentiation of NIE-115 cells caused an increase in the levels of rbB, rbC, and rbD RNAs. Differentiation by nerve growth factor caused an increase in levels of all four genes in PC-12. Our data give further support for the assignment of rbA, rbB, and rbC/rbD gene products as components of P-, N-, and L-type Ca2+ channels, respectively.

scholarly journals Processing and secretion of human carboxypeptidase E by C6 glioma cells

1991 ◽  
Vol 280 (3) ◽  
pp. 695-701 ◽  
Author(s):  
E Manser ◽  
D Fernandez ◽  
L Lim
Keyword(s):  
Glioma Cell Line ◽  
C6 Glioma Cells ◽  
Soluble Form ◽  
Multiple Forms ◽  
Post Translational Modification ◽  
Rat Glioma ◽  
Carboxypeptidase E ◽  
Metallothionein Promoter ◽  
Murine Sarcoma Virus ◽  
Membrane Bound

Carboxypeptidase E (CPE) catalyses the removal of C-terminal basic amino acids and is implicated in the processing of peptides and hormones in secretory vesicles. The enzyme has been isolated in multiple forms from both soluble and membrane-bound compartments, and has been demonstrated to be co-secreted with peptides from pancreatic and adrenal cells. To address the question regarding the origin of the multiple forms of the enzyme, we have transfected a construct containing the cDNA for human CPE under the control of the murine-sarcoma-virus enhancer and metallothionein promoter into the C6 rat glioma cell line, which itself has extremely low levels of CPE expression. Within transfectants, multiple forms of the enzyme have been detected by Western blotting, and their sizes are comparable with that in pituitary. Fractionation of the intracellular forms of CPE with Triton X-114 at various pH values indicates that the membrane-bound, but not the soluble, form is amphipathic; this difference probably arises from post-translational modification of the enzyme. Interestingly, the secreted enzyme is found to have the amphipathic characteristics of the membrane-bound form. By modification of the cDNA sequence, enzyme lacking N-terminal and C-terminal domains have been expressed: deletion of the ‘pro’ region of CPE, leading to loss of the signal cleavage site, gives a more hydrophobic but active enzyme which is not efficiently secreted from the cell; deletion of a C-terminal region gives a secreted form of the enzyme which no longer exhibits amphipathic behaviour. Our data indicate that a single mRNA species can give rise to the multiple forms of CPE enzyme that have been identified and that the C6 cells are able to carry out the intracellular processing events to generate these forms.

scholarly journals The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase

2004 ◽  
Vol 381 (3) ◽  
pp. 831-840 ◽  
Author(s):  
Sheng LI ◽  
Jon A. FRIESEN ◽  
Hong FEI ◽  
Xiang DING ◽  
David W. BORST
Keyword(s):  
Amino Acid ◽  
Homarus Americanus ◽  
Class I ◽  
Soluble Form ◽  
Molecular Characteristics ◽  
Mrna Levels ◽  
Acid Protein ◽  
Membrane Bound ◽  
Coa Reductase ◽  
Amino Acid Protein

In a previous study [Li, Wagner, Friesen and Borst (2003) Gen. Comp. Endocrinol. 134, 147–155], we showed that the MO (mandibular organ) of the lobster Homarus americanus has high levels of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and that most (approx. 75%) of the enzyme activity is soluble. In the present study, we report the biochemical and molecular characteristics of this enzyme. HMGR had two forms in the MO: a more abundant soluble form (66 kDa) and a less abundant membrane-bound form (72 kDa). Two cDNAs for HMGR were isolated from the MO. A 2.6-kb cDNA encoded HMGR1, a 599-amino-acid protein (63 kDa), and a 3.2-kb cDNA encoded HMGR2, a 655-amino-acid protein (69 kDa). These two cDNAs had identical 3′-ends and appeared to be products of a single gene. The deduced amino acid sequences of these two proteins revealed a high degree of similarity to other class I HMGRs. Hydropathy plots indicated that the N-terminus of HMGR1 lacked a transmembrane region and HMGR2 had a single transmembrane segment. Recombinant HMGR1 expressed in Sf9 insect cells was soluble and had kinetic characteristics similar to native HMGR from the MO. Treatment with phosphatase did not affect HMGR activity, consistent with the observation that neither HMGR1 nor HMGR2 has a serine at position 490 or 546, the position of a conserved phosphorylation site found in class I HMGR from higher eukaryotes. Other lobster tissues (i.e. midgut, brain and muscles) had low HMGR activities and mRNA levels. MO with higher HMGR activities had higher HMGR mRNA levels, implying that HMGR is regulated, in part, at the transcription level.

scholarly journals β-Cell Lines Derived from Transgenic Cpefat/Cpefat Mice Are Defective in Carboxypeptidase E and Proinsulin Processing*

Endocrinology ◽  
1997 ◽  
Vol 138 (11) ◽  
pp. 4883-4892 ◽  
Author(s):  
Oleg Varlamov ◽  
Lloyd D. Fricker ◽  
Hisasi Furukawa ◽  
Donald F. Steiner ◽  
Stephen H. Langley ◽  
...  
Keyword(s):  
Cell Lines ◽  
Β Cell ◽  
Carboxypeptidase E ◽  
Proinsulin Processing

Analysis of the Anterior Secretory Cells of Onchidohs Muricata (Nudibranchia)

1981 ◽  
Vol 39 ◽  
pp. 614-615
Author(s):  
Roy Skidmore
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Granules ◽  
Golgi Body ◽  
The Body ◽  
Secretory Cells ◽  
Secretory Vesicles ◽  
High Magnification ◽  
Large Numbers ◽  
Membrane Bound ◽  
Sole Of The Foot

The long-necked secretory cells in Onchidoris muricata are distributed in the anterior sole of the foot. These cells are interspersed among ciliated columnar and conical cells as well as short-necked secretory gland cells. The long-necked cells contribute a significant amount of mucoid materials to the slime on which the nudibranch travels. The body of these cells is found in the subepidermal tissues. A long process extends across the basal lamina and in between cells of the epidermis to the surface of the foot. The secretory granules travel along the process and their contents are expelled by exocytosis at the foot surface.The contents of the cell body include the nucleus, some endoplasmic reticulum, and an extensive Golgi body with large numbers of secretory vesicles (Fig. 1). The secretory vesicles are membrane bound and contain a fibrillar matrix. At high magnification the similarity of the contents in the Golgi saccules and the secretory vesicles becomes apparent (Fig. 2).

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 953
Author(s):  
Chuan Xu ◽  
Annie Wang ◽  
Ke Geng ◽  
William Honnen ◽  
Xuening Wang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Viral Entry ◽  
Broad Spectrum ◽  
A549 Cells ◽  
Cell Types ◽  
Pseudotyped Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Human Immunodeficiency Viruses ◽  
Tyrosine Protein Kinase ◽  
Overexpressing Cell

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.

scholarly journals YB-1 Is Altered in Pregnancy-Associated Disorders and Affects Trophoblast in Vitro Properties via Alternation of Multiple Molecular Traits

2021 ◽  
Vol 22 (13) ◽  
pp. 7226
Author(s):  
Violeta Stojanovska ◽  
Aneri Shah ◽  
Katja Woidacki ◽  
Florence Fischer ◽  
Mario Bauer ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Progression ◽  
Cell Function ◽  
Trophoblast Cell ◽  
Mrna Levels ◽  
Trophoblast Cells ◽  
Invasion And Migration ◽  
And Migration ◽  
Apoptosis And Inflammation

Cold shock Y-box binding protein-1 (YB-1) coordinates several molecular processes between the nucleus and the cytoplasm and plays a crucial role in cell function. Moreover, it is involved in cancer progression, invasion, and metastasis. As trophoblast cells share similar characteristics with cancer cells, we hypothesized that YB-1 might also be necessary for trophoblast functionality. In samples of patients with intrauterine growth restriction, YB-1 mRNA levels were decreased, while they were increased in preeclampsia and unchanged in spontaneous abortions when compared to normal pregnant controls. Studies with overexpression and downregulation of YB-1 were performed to assess the key trophoblast processes in two trophoblast cell lines HTR8/SVneo and JEG3. Overexpression of YB-1 or exposure of trophoblast cells to recombinant YB-1 caused enhanced proliferation, while knockdown of YB-1 lead to proliferative disadvantage in JEG3 or HTR8/SVneo cells. The invasion and migration properties were affected at different degrees among the trophoblast cell lines. Trophoblast expression of genes mediating migration, invasion, apoptosis, and inflammation was altered upon YB-1 downregulation. Moreover, IL-6 secretion was excessively increased in HTR8/SVneo. Ultimately, YB-1 directly binds to NF-κB enhancer mark in HTR8/SVneo cells. Our data show that YB-1 protein is important for trophoblast cell functioning and, when downregulated, leads to trophoblast disadvantage that at least in part is mediated by NF-κB.

scholarly journals Retraction Note: Flavonoids modulate multidrug resistance through wnt signaling in P-glycoprotein overexpressing cell lines

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
S. Mohana ◽  
M. Ganesan ◽  
N. Rajendra Prasad ◽  
D. Ananthakrishnan ◽  
D. Velmurugan
Keyword(s):  
Multidrug Resistance ◽  
Wnt Signaling ◽  
Cell Lines ◽  
P Glycoprotein ◽  
Overexpressing Cell

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