scholarly journals Internalization of the opioid growth factor, [Met5]-enkephalin, is dependent on clathrin-mediated endocytosis for downregulation of cell proliferation

2010 ◽  
Vol 299 (3) ◽  
pp. R774-R785 ◽  
Author(s):  
Fan Cheng ◽  
Patricia J. McLaughlin ◽  
William A. Banks ◽  
Ian S. Zagon
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Wound Repair ◽  
Opioid Antagonist ◽  
Live Cells ◽  
Inhibitory Peptide ◽  
African Green Monkey Kidney ◽  
Initial Exposure ◽  
Opioid Growth Factor

The opioid growth factor (OGF; [Met5]-enkephalin), a constitutively expressed and tonically active inhibitory peptide, interacts with the OGF receptor (OGFr) to form an endogenous growth-regulating pathway in homeostasis. Amplification of OGF-OGFr interfacing in animal and clinical studies depresses development, neoplasia, angiogenesis, and immunity. Disruption of the OGF-OGFr axis accelerates cell proliferation and has been particularly important in wound repair. To investigate how OGF enters cells, OGF was labeled with 5,6-tetramethylrhodamine OGF (RhoOGF) to study its uptake in live cells. African green monkey kidney cells (COS-7) incubated with RhoOGF exhibited a temperature-dependent course of entry, being internalized at 37°C but not at 4°C. RhoOGF was detected in the cytoplasm 15 min after initial exposure, observed in both cytoplasm and nucleus within 30 min, and remained in the cells for as long as 5 h. A 100-fold excess of OGF or the opioid antagonist naltrexone, but not other opioid ligands (some selective for classic opioid receptors), markedly reduced entry of RhoOGF into cells. RhoOGF was functional because DNA synthesis in cells incubated with RhoOGF (10−5 to 10−8 M) was decreased 24–36%, and was comparable to cells treated with unlabeled OGF (reductions of 26–39%). OGF internalization was dependent on clathrin-mediated endocytosis, with addition of clathrin siRNA diminishing the uptake of RhoOGF and upregulating DNA synthesis. RhoOGF clathrin-mediated endocytosis was unrelated to endosomal or Golgi pathways. Taken together, these results suggest that OGF enters cells by active transport in a saturable manner that requires clathrin-mediated endocytosis.

Opioid growth factor is present in human and mouse gastrointestinal tract and inhibits DNA synthesis

1997 ◽  
Vol 272 (4) ◽  
pp. R1094-R1104
Author(s):  
I. S. Zagon ◽  
Y. Wu ◽  
P. J. McLaughlin
Keyword(s):  
Circadian Rhythm ◽  
Growth Factor ◽  
Epithelial Cells ◽  
Dna Synthesis ◽  
Opioid Antagonist ◽  
Receptor Interaction ◽  
Esophageal Epithelium ◽  
Endogenous Opioid ◽  
Endogenous Opioid Peptide ◽  
Opioid Growth Factor

Native opioid peptides serve as growth factors in a number of normal and neoplastic cells and tissues. This study investigated the influence of opioids on circadian rhythm-dependent DNA synthesis in mouse esophagus during homeostatic renewal. In contrast to a labeling index (LI) of 24.0% at 0630 and 5.5% at 1600, disruption of opioid-receptor interaction by the potent opioid antagonist naltrexone hydrochloride (NTX; 10 mg/kg) in mice resulted in an elevation of 49% in DNA synthesis of esophageal epithelial cells at 1600, but had no effect at 0630. Mice subjected to [Met5]enkephalin (1 mg/kg) had an LI that was decreased 23% from control levels at 0630, but was unaffected at 1600. This decrease in DNA synthesis was blocked by concomitant administration of naloxone (10 mg/kg); naloxone alone had no influence on cell replicative processes. In tissue culture studies, NTX and OGF markedly increased and decreased, respectively, the LI from control values. Both opioid growth factor (OGF) and its receptor, zeta, were detected in all but the cornified layer of mouse esophageal epithelium and in the epithelial cells of the stomach and small and large intestines. In addition, both peptide and receptor were observed in the basal and suprabasal cells of human esophageal epithelium. These results indicate that an endogenous opioid peptide (OGF) and its receptor (zeta) reside in gastrointestinal epithelium and play a role in cellular renewal processes in a tonically inhibitory, direct, and circadian rhythm-dependent fashion.

Targeted overexpression of OGFr in epithelium of transgenic mice suppresses cell proliferation and impairs full-thickness wound closure

2012 ◽  
Vol 302 (9) ◽  
pp. R1084-R1090 ◽  
Author(s):  
Patricia J. McLaughlin ◽  
Cara L. Keiper ◽  
Michael F. Verderame ◽  
Ian S. Zagon
Keyword(s):  
Cell Proliferation ◽  
Transgenic Mice ◽  
Dna Synthesis ◽  
Wound Repair ◽  
Fold Increase ◽  
Binding Activity ◽  
Feedback Mechanism ◽  
Opioid Antagonist ◽  
Full Thickness ◽  
Wild Type

The opioid growth factor (OGF) and its receptor, OGFr, play a regulatory role in cell proliferation, and maintain homeostasis through a tonically active negative feedback mechanism. To directly evaluate the repercussion of increased OGFr expression and consequent gain-of-function in epithelium, bovine keratin 5 promoter elements were used to direct the expression of OGFr to skin in a tetracycline-regulated manner. Three founder lines overexpressing OGFr (OGFrTG/K5-tTA) were established. Evidence for increased OGFr in the epithelium included a three-fold increase in OGFr binding activity, as well as significant increases in OGFr protein, as monitored by semiquantitative immunohistochemistry. DNA synthesis in target epithelium, including cornea, tongue, and skin of transgenic mice was decreased 41% to 80% from wild-type littermates; the liver, a nonepithelial organ, was not altered. Decreased DNA synthesis in corneal epithelium induced by transgenic expression of OGFr was further reduced by treatment with exogenous OGF but reversed by exposure to the opioid antagonist, naloxone. The number of cell layers in both epidermis and cornea of OGFrTG/K5-tTA animals was reduced nearly 45% from wild-type mice. Full-thickness wounds in mice overexpressing OGFr healed 37% to 75% slower than wild-type littermates. These data demonstrate for the first time that stable genetic amplification of OGFr downregulates homeostatic cell proliferation, as well as pathophysiological processes with respect to wound repair. These mice also can serve as a valuable model to dissect the mechanism of OGF-OGFr action and may be important in understanding the etiology, pathogenesis, and treatment of epithelium-related diseases.

Opioid growth factor inhibits DNA synthesis in mouse tongue epithelium in a circadian rhythm-dependent manner

1994 ◽  
Vol 267 (3) ◽  
pp. R645-R652 ◽  
Author(s):  
I. S. Zagon ◽  
Y. Wu ◽  
P. J. McLaughlin
Keyword(s):  
Circadian Rhythm ◽  
Growth Factor ◽  
Dna Synthesis ◽  
Opioid Peptide ◽  
Endogenous Opioids ◽  
Opioid Antagonist ◽  
Dependent Manner ◽  
Endogenous Opioid ◽  
Endogenous Opioid Peptide ◽  
Opioid Growth Factor

In addition to neuromodulation, endogenous opioids also serve as growth factors. To investigate the involvement of the naturally occurring opioid peptide [Met5]enkephalin [termed opioid growth factor (OGF)] in the renewal of epithelium, adult mice were given systemic injections of OGF (1 mg/kg) and examined 2 h later at 0700 or 1700 h. DNA synthesis in the tongue was investigated using [3H]thymidine and autoradiography. OGF depressed DNA synthesis of the basal epithelial cells in the tip, and dorsal and ventral surfaces of the tongue (42-44% of control levels) only at 0700 h. This decrease in DNA synthesis was blocked by concomitant administration of the opioid antagonist naloxone (10 mg/kg); naloxone alone had no influence on cell replicative processes. Both OGF and its receptor, zeta (zeta), were detected in the stratified squamous epithelium of the ventral and dorsal surfaces of the tongue by immunocytochemistry. Photodensitometric measurements of immunocytochemical preparations revealed almost twofold more OGF and zeta-receptor immunoreactivity at 1700 h than at 0700 h. These results indicate that an endogenous opioid peptide and its receptor are present and govern cellular renewal processes in the tongue and regulate DNA synthesis in a circadian rhythm-dependent fashion.

Passive diffusion of naltrexone into human and animal cells and upregulation of cell proliferation

2009 ◽  
Vol 297 (3) ◽  
pp. R844-R852 ◽  
Author(s):  
Fan Cheng ◽  
Patricia J. McLaughlin ◽  
William A. Banks ◽  
Ian S. Zagon
Keyword(s):  
Cell Proliferation ◽  
Spatial Distribution ◽  
Dna Synthesis ◽  
Cell Line ◽  
Pancreatic Cancer Cell Line ◽  
Passive Diffusion ◽  
Human Pancreatic Cancer ◽  
Opioid Antagonist ◽  
Cytoplasmic Staining ◽  
African Green Monkey Kidney

Naltrexone (NTX) is a potent opioid antagonist that promotes cell proliferation by upregulating DNA synthesis through displacement of the tonically active inhibitory peptide, opioid growth factor (OGF) from its receptor (OGFr). To investigate how NTX enters cells, NTX was fluorescently labeled [1-( N)-fluoresceinyl NTX thiosemicarbazone; FNTX] to study its uptake by living cultured cells. When human head and neck squamous cell carcinoma cell line (SCC-1) was incubated with FNTX for as little as 1 min, cells displayed nuclear and cytoplasmic staining of FNTX as determined by fluorescent deconvolution microscopy, with enrichment of fluorescent signal in the nucleus and nucleolus. The same temporal-spatial distribution of FNTX was detected in a human pancreatic cancer cell line (MIA PaCa-2), African green monkey kidney cell line (COS-7), and human mesenchymal stem cells (hMSCs). FNTX remained in cells for as long as 48 h. FNTX was internalized in SCC-1 cells when incubation occurred at 4°C, with the signal being comparable to that recorded at 37°C. A 100-fold excess of NTX or a variety of other opioid ligands did not alter the temporal-spatial distribution of FNTX. Neither fluorescein-labeled dextran nor fluorescein alone entered the cells. To study the effect of FNTX on DNA synthesis, cells incubated with FNTX at concentrations ranging from 10−5 to 10−8 M had a 5-bromo-2′-deoxyuridine index that was 39–82% greater than for vehicle-treated cells and was comparable to that of unlabeled NTX (37–70%). Taken together, these results suggested that NTX enters cells by passive diffusion in a nonsaturable manner.

scholarly journals Regulation of Tenon's Capsule Fibroblast Cell Proliferation by the Opioid Growth Factor and the Opioid Growth Factor Receptor Axis

2010 ◽  
Vol 51 (10) ◽  
pp. 5054 ◽  
Author(s):  
Matthew S. Klocek ◽  
Joseph W. Sassani ◽  
Renee N. Donahue ◽  
Patricia J. McLaughlin ◽  
Ian S. Zagon
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Growth Factor Receptor ◽  
Fibroblast Cell ◽  
Opioid Growth Factor ◽  
Tenon’S Capsule ◽  
Tenon's Capsule

scholarly journals Effects of Local Opioid Antagonist on Diabetic Fracture Rat Model

2018 ◽  
Vol 3 (3) ◽  
pp. 2473011418S0017
Author(s):  
Jarrett D. Cain ◽  
Michelle Titunick ◽  
Patricia McLaughlin ◽  
Ian Zagon
Keyword(s):  
Growth Factor ◽  
Fracture Healing ◽  
Bone Healing ◽  
Calcium Sulfate ◽  
Bone Fractures ◽  
Diabetic Rats ◽  
Receptor Expression ◽  
Opioid Antagonist ◽  
Diabetic Patients ◽  
Opioid Growth Factor

Category: Diabetes Introduction/Purpose: Complications associated with the diabetes include increased incidence of fracture healing, delayed fracture healing, delayed osteoblasts cell replication, decreased angiogenesis, migration and/or osteoblast cell differentiation. The cellular events involved in bone healing are adversely affected by diabetes; however, can be modulated by the Opioid Growth Factor (OGF)–OGF receptor (OGFr) is an inhibitory peptide that downregulates DNA synthesis in a tissue nonspecific manner. Diabetes is associated with elevated serum levels of OGF and dysregulation of the OGFr leading to multiple complications related to healing, sensitivity, and regeneration. This study explores the presence and function of the OGF-OGFr axis in bone tissue from type 1 diabetic rats examining intact and fractured femurs during early phases of the repair process Methods: Seven-week-old Sprague Dawley rats were injected with streptozotocin (40mg/kg i.p.) to induce T1D; other rats received buffer only and served as controls. After one month, hyperglycemia rats underwent surgery to produce a fracture at the distal third of the femur. Four diabetic rats received opioid antagoinist (naltrexone) and calcium sulfate and all remaining rats received calcium sulfate with water only. X-rays were taken immediately after surgery and after rats were euthanized on post-surgery; femur and tibia were collected for protein isolation, western blot analysis along with frozen or paraffin-embedded for histological analysis Results: Immunofluorescence indicated approximately 90% increase in opioid growth factor receptor expression in diabetic femurs compared to age-matched normal femurs. Western Blotting also suggested an increase in the receptor protein in diabetic bones relative to normal bone. TRAP staining for osteoclasts was greater in control and opioid antagonist-treated diabetic fractures when compared to the number of osteoclasts in vehicle-treated diabetic fractured femurs. Safranin O stained sections revealed approximately more bone in opioid growth receptor antagonist-treated diabetic bone fractures than in vehicle-treated bone fractures Conclusion: These data support our hypothesis that expression levels of OGFr are dysregulated in the bone of diabetic patients leading to complications in bone healing. Moreover, modulation of the OGF-OGFr pathway with receptor antagonists restored some aspects of bone healing. With further study, these preliminary results support the role of the OGF-OGFr axis in treatment of diabetic bone healing. New therapies to target dysregulation of the OGF-OGFr regulatory pathway in diabetes would provide a safe and effective disease-modifying treatment for delayed bone healing.

Effects of epidermal growth factor on diamine oxidase expression and cell growth in Caco-2 cells

1991 ◽  
Vol 261 (4) ◽  
pp. G669-G676 ◽  
Author(s):  
B. Daniele ◽  
A. Quaroni
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Dna Synthesis ◽  
Intestinal Mucosa ◽  
Diamine Oxidase ◽  
Small Intestinal Mucosa ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Epidermal Growth

To investigate the role of diamine oxidase (DAO) in the intestinal mucosa, we compared its expression with cell proliferation and differentiation in the human colon carcinoma cell line Caco-2. DAO synthesis was evaluated in subconfluent and confluent cultures and in the presence of epidermal growth factor (EGF), a polypeptide hormone known to have specific trophic effects on the small intestinal mucosa. EGF stimulated DNA synthesis, significantly increased cellular DAO activity and the amount of enzyme secreted into the culture medium, but decreased expression of dipeptidyl peptidase IV, a marker of cell differentiation in confluent Caco-2 cells. Immunoprecipitation of DAO from cells labeled metabolically with [35S]methionine failed to demonstrate an increased enzyme synthesis in EGF-treated cells, suggesting that this hormone acted primarily at a posttranslational level by reducing DAO degradation before intracellular storage or secretion. A possible relationship between changes in cellular DAO activity and cell proliferation was also investigated by using aminoguanidine, a specific and potent DAO inhibitor. Although DAO activity was markedly suppressed, aminoguanidine had no significant effects on the rate of DNA synthesis. These results demonstrated that in Caco-2 cells EGF stimulated DNA synthesis and DAO expression; however, cell proliferation and differentiation were not correlated with the levels of cellular DAO, suggesting that this enzyme does not play a major role in the regulation of intestinal epithelial cell turnover.

Sign in / Sign up

Export Citation Format

Share Document