Reduced Stem Cell Factor Links Smooth Myopathy and Loss of Interstitial Cells of Cajal in Murine Diabetic Gastroparesis
2006; Elsevier BV; Volume: 130; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2005.12.027
ISSN1528-0012
AutoresViktor J. Horváth, Harsha Vittal, Andrea Lörincz, Hui Chen, Graça Almeida‐Porada, Doug Redelman, Tamás Ördög,
Tópico(s)Caveolin-1 and cellular processes
ResumoBackground & Aims: Diabetic gastroparesis involves neuropathy, myopathy, and depletion of interstitial cells of Cajal (ICC), which may cause dysrhythmias and impaired neural control. Most murine gastric ICC depend on stem cell factor (SCF) signaling but can also be maintained with insulin or insulin-like growth factor-I (IGF-I). We investigated whether SCF could mediate the actions of insulin and IGF-I. Methods: Expression of insulin receptor, IGF-I receptor, and SCF was studied in gastric muscles and purified ICC by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The effects of insulin/IGF-I deficiency on SCF, ICC, smooth muscle, and neurons were investigated in nonobese diabetic mice and organotypic cultures by immunohistochemistry, microarrays, and/or quantitative RT-PCR. ICC in organotypic cultures were also studied after immunoneutralization of endogenous SCF. Results: Insulin and IGF-I receptors were detected in smooth-muscle cells and myenteric neurons but not in ICC. Cell-surface expression of SCF was only found in smooth-muscle cells. ICC depletion in diabetes was accompanied by smooth-muscle atrophy and reduced SCF, whereas neuron-specific gene expression remained unchanged. In organotypic cultures, prevention of ICC loss by insulin or IGF-I was paralleled by rescue of smooth-muscle cells and SCF expression but not of myenteric neurons. Immunoneutralization of endogenous SCF caused ICC depletion closely resembling that elicited by insulin/IGF-I deficiency. Conclusions: Reduced insulin/IGF-I signaling in diabetes may lead to ICC depletion and its consequences by causing smooth-muscle atrophy and reduced SCF production. Thus, myopathy may play a more central role in diabetic gastroenteropathies than previously recognized. Background & Aims: Diabetic gastroparesis involves neuropathy, myopathy, and depletion of interstitial cells of Cajal (ICC), which may cause dysrhythmias and impaired neural control. Most murine gastric ICC depend on stem cell factor (SCF) signaling but can also be maintained with insulin or insulin-like growth factor-I (IGF-I). We investigated whether SCF could mediate the actions of insulin and IGF-I. Methods: Expression of insulin receptor, IGF-I receptor, and SCF was studied in gastric muscles and purified ICC by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The effects of insulin/IGF-I deficiency on SCF, ICC, smooth muscle, and neurons were investigated in nonobese diabetic mice and organotypic cultures by immunohistochemistry, microarrays, and/or quantitative RT-PCR. ICC in organotypic cultures were also studied after immunoneutralization of endogenous SCF. Results: Insulin and IGF-I receptors were detected in smooth-muscle cells and myenteric neurons but not in ICC. Cell-surface expression of SCF was only found in smooth-muscle cells. ICC depletion in diabetes was accompanied by smooth-muscle atrophy and reduced SCF, whereas neuron-specific gene expression remained unchanged. In organotypic cultures, prevention of ICC loss by insulin or IGF-I was paralleled by rescue of smooth-muscle cells and SCF expression but not of myenteric neurons. Immunoneutralization of endogenous SCF caused ICC depletion closely resembling that elicited by insulin/IGF-I deficiency. Conclusions: Reduced insulin/IGF-I signaling in diabetes may lead to ICC depletion and its consequences by causing smooth-muscle atrophy and reduced SCF production. Thus, myopathy may play a more central role in diabetic gastroenteropathies than previously recognized. Gastroparesis, defined as gastric retention without mechanical obstruction, and gastropathy, the symptom complex typically associated with gastroparesis, occur in 25%–55% of patients with long-standing type 1 or type 2 diabetes.1Parkman H.P. Hasler W.L. Fisher R.E. American Gastroenterological Association technical review on the diagnosis and treatment of gastroparesis.Gastroenterology. 2004; 127: 1592-1622Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 2Camilleri M. Advances in diabetic gastroparesis.Rev Gastroenterol Disord. 2002; 2: 47-56PubMed Google Scholar Symptoms may include nausea and vomiting, which may be self-limiting, recurrent, or unrelenting; bloating; early satiety; abdominal discomfort; and pain (dyspepsia).1Parkman H.P. Hasler W.L. Fisher R.E. 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Firstly, ICC forming 2-dimensional networks in the myenteric region generate electrical slow waves that govern phasic contractile activity and provide a pathway for slow wave propagation required for peristalsis.16Dickens E.J. Hirst G.D.S. Tomita T. Identification of rhythmically active cells in guinea-pig stomach.J Physiol (Lond). 1999; 514: 515-531Crossref Scopus (270) Google Scholar, 17Ördög T. Ward S.M. Sanders K.M. Interstitial cells of Cajal generate electrical slow waves in the murine stomach.J Physiol (Lond). 1999; 518: 257-269Crossref Scopus (199) Google Scholar, 18Ördög T. Baldo M. Danko R. Sanders K.M. Plasticity of electrical pacemaking by interstitial cells of Cajal and gastric dysrhythmias in W/Wv mutant mice.Gastroenterology. 2002; 123: 2028-2040Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar Disruption of these networks has been shown to result in electrical dysrhythmias (bradygastrias, tachyarrhythmias, and pacemaker dissociation) and consequent delayed gastric emptying that are considered hallmarks of gastroparesis.12Ördög T. Takayama I. Cheung W.K.T. Ward S.M. Sanders K.M. Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis.Diabetes. 2000; 49: 1731-1739Crossref PubMed Scopus (274) Google Scholar, 17Ördög T. Ward S.M. Sanders K.M. Interstitial cells of Cajal generate electrical slow waves in the murine stomach.J Physiol (Lond). 1999; 518: 257-269Crossref Scopus (199) Google Scholar, 18Ördög T. Baldo M. Danko R. Sanders K.M. 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Selective loss of vagal intramuscular mechanoreceptors in mice mutant for steel factor, the c-Kit receptor ligand.Anat Embryol. 2002; 205: 325-342Crossref PubMed Scopus (66) Google Scholar The loss of these cells can mimic autonomic neuronal dysfunction (eg, by causing reduced receptive and sphincter relaxation12Ördög T. Takayama I. Cheung W.K.T. Ward S.M. Sanders K.M. Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis.Diabetes. 2000; 49: 1731-1739Crossref PubMed Scopus (274) Google Scholar, 19Ward S.M. Sanders K.M. Interstitial cells of Cajal primary targets of enteric motor innervation.Anat Rec. 2001; 262: 125-135Crossref PubMed Scopus (165) Google Scholar, 20Beckett E.A.H. Horiguchi K. Khoyi M. Sanders K.M. Ward S.M. Loss of enteric motor neurotransmission in the gastric fundus of Sl/Sld mice.J Physiol (Lond). 2002; 543: 871-887Crossref Scopus (121) Google Scholar and altered sensory functions21Fox E.A. Phillips R.J. Byerly M.S. Baronowsky E.A. Chi M.M. Powley T.L. Selective loss of vagal intramuscular mechanoreceptors in mice mutant for steel factor, the c-Kit receptor ligand.Anat Embryol. 2002; 205: 325-342Crossref PubMed Scopus (66) Google Scholar). Thus, disruptions of ICC networks in diabetes can potentially affect all aspects of gastric neuromuscular function and could represent an important therapeutic target. The mechanism of diabetes-associated loss of ICC is not fully understood. It is well established that ICC survival and function depend on the activation of c-Kit, a receptor tyrosine kinase integral to ICC, by stem cell factor (SCF, Kit ligand) produced locally within the tunica muscularis.17Ördög T. Ward S.M. Sanders K.M. Interstitial cells of Cajal generate electrical slow waves in the murine stomach.J Physiol (Lond). 1999; 518: 257-269Crossref Scopus (199) Google Scholar, 22Torihashi S. Yoshida H. Nishikawa S. Kunisada T. Sanders K.M. Eneteric neurons express Steel factor-lacZ transgene in the murine gastrointestinal tract.Brain Res. 1996; 738: 323-328Crossref PubMed Scopus (46) Google Scholar, 23Ward S.M. Ördög T. Bayguinov J.R. Horowitz B. Epperson A. Shen L. Westphal H. Sanders K.M. Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves.Gastroenterology. 1999; 117: 584-594Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 24Wu J.J. Rothman T.P. Gershon M.D. Development of the interstitial cells of Cajal origin, Kit dependence and neuronal and nonneuronal sources of Kit ligand.J Neurosci Res. 2000; 59: 384-401Crossref PubMed Scopus (136) Google Scholar, 25Huizinga J.D. Berezin I. Sircar K. Hewlett B. Donnelly G. Bercik P. Ross C. Algoufi T. Fitzgerald P. Der T. Riddell R.H. Collins S.M. Jacobson K. Development of interstitial cells of Cajal in a full-term infant without an enteric nervous system.Gastroenterology. 2001; 120: 561-567Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar By using organotypic cultures, which permit the independent manipulation of glucose, insulin, and insulin-like growth factor-I (IGF-I) levels for the time typically required for gastroparesis and ICC loss to develop in diabetic NOD mice (up to 2–3 months),12Ördög T. Takayama I. Cheung W.K.T. Ward S.M. Sanders K.M. Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis.Diabetes. 2000; 49: 1731-1739Crossref PubMed Scopus (274) Google Scholar we have recently found that gastric ICC are resistant to hyperglycemia but depend on insulin or IGF-I for survival and function.26Horváth V.J. Vittal H. Ördög T. Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetes mellitus-associated depletion of interstitial cells of Cajal in the murine stomach.Diabetes. 2005; 54: 1528-1533Crossref PubMed Scopus (86) Google Scholar Because these experiments were performed in gastric muscles placed in culture, the effects of insulin and IGF-I may not have been mediated by specific receptors expressed by ICC. Therefore, the goal of the present study was to examine whether ICC are direct targets of insulin and IGF-I signaling or whether the effects of these cytokines are mediated by SCF, the only established growth factor for ICC. Mice were maintained and the experiments performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the Institutional Animal Use and Care Committee at the University of Nevada, Reno. Young adult and 9–18-day-old BALB/c mice were obtained from our institutional breeding program. Female NOD mice (5–6-weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME). Diagnosis and monitoring of diabetes in NOD mice were performed by monitoring glycosuria as described.12Ördög T. Takayama I. Cheung W.K.T. Ward S.M. Sanders K.M. Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis.Diabetes. 2000; 49: 1731-1739Crossref PubMed Scopus (274) Google Scholar Diabetic mice were used when signs of gastrointestinal (distended abdomen), and other complications (impaired locomotion and vision) became evident by inspection.12Ördög T. Takayama I. Cheung W.K.T. Ward S.M. Sanders K.M. Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis.Diabetes. 2000; 49: 1731-1739Crossref PubMed Scopus (274) Google Scholar Age-matched nondiabetic littermates were used as controls.12Ördög T. Takayama I. Cheung W.K.T. Ward S.M. Sanders K.M. Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis.Diabetes. 2000; 49: 1731-1739Crossref PubMed Scopus (274) Google Scholar The animals were anesthetized with isoflurane (Baxter Healthcare, Deerfield, IL) before decapitation. Intact muscle layers representing the entire gastric corpus and antrum were prepared by peeling away the mucosa and submucosa. Some anatomically defined gastric corpus + antrum muscles were incubated at 45°C until weight equilibrium to obtain dry weights. Intact, continuous corpus and antrum muscles from juvenile BALB/c mice were cultured in normoglycemic (5.55 mmol/L) Medium 199 (Sigma, St Louis, MO) as described.17Ördög T. Ward S.M. Sanders K.M. Interstitial cells of Cajal generate electrical slow waves in the murine stomach.J Physiol (Lond). 1999; 518: 257-269Crossref Scopus (199) Google Scholar, 26Horváth V.J. Vittal H. Ördög T. Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetes mellitus-associated depletion of interstitial cells of Cajal in the murine stomach.Diabetes. 2005; 54: 1528-1533Crossref PubMed Scopus (86) Google Scholar Supplements included d-glucose (55.5 mmol/L), bovine insulin (5 μg/mL; Invitrogen, Carlsbad, CA), murine IGF-I (100 ng/mL, Sigma), or rabbit polyclonal anti-SCF antibodies (H-189, 2 μg/mL; Santa Cruz Biotechnology, Santa Cruz, CA). Before use, the anti-SCF antibodies were extensively dialyzed with 0.01 N phosphate-buffered saline (3 × 2 L, 24 hours at 4°C). All treatments were maintained throughout the entire experiment. Levels of the supplements were kept constant by changing the culture media every 48 hours.26Horváth V.J. Vittal H. Ördög T. Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetes mellitus-associated depletion of interstitial cells of Cajal in the murine stomach.Diabetes. 2005; 54: 1528-1533Crossref PubMed Scopus (86) Google Scholar Cultured and freshly dissected tissues were processed and immunostained as described.17Ördög T. Ward S.M. Sanders K.M. Interstitial cells of Cajal generate electrical slow waves in the murine stomach.J Physiol (Lond). 1999; 518: 257-269Crossref Scopus (199) Google Scholar, 18Ördög T. Baldo M. Danko R. Sanders K.M. Plasticity of electrical pacemaking by interstitial cells of Cajal and gastric dysrhythmias in W/Wv mutant mice.Gastroenterology. 2002; 123: 2028-2040Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 26Horváth V.J. Vittal H. Ördög T. Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetes mellitus-associated depletion of interstitial cells of Cajal in the murine stomach.Diabetes. 2005; 54: 1528-1533Crossref PubMed Scopus (86) Google Scholar The following primary antibodies were used: rat IgG2b monoclonal anti–c-Kit clone ACK2 (5 μg/mL), rabbit polyclonal anti-insulin receptor α, anti–IGF-I receptor α, anti-SCF, goat polyclonal anti-insulin receptor β (Santa Cruz Biotechnology; 2 μg/mL), and rabbit polyclonal anti-PGP 9.5 (Biogenesis, Brentwood, NH; 1:200). Secondary antibodies were conjugated with either Alexa Fluor 488, 594, or Texas red (Molecular Probes, Eugene, OR). Specificity was verified by omitting the primary or secondary antibodies and by preabsorption with the appropriate blocking peptide. Live immunolabeling was performed by incubating gastric and jejunal tunica muscularis tissues with antibodies against SCF (2 μg/mL) and neural cell adhesion molecule (CD56; clone: H28.123; 2 μg/mL; Beckman Coulter, Fullerton, CA) at 4°C for 3 hours. Secondary antibodies were applied after fixation with 4% paraformaldehyde-saline. Confocal microscopy was performed using a Bio-Rad MRC 600 (Hercules, CA) or a Zeiss LSM 510 META confocal system (Carl Zeiss Microimaging, Thornwood, NY).27Ördög T. Redelman D. Miller L.J. Horváth V.J. Zhong Q. Almeida-Porada G. Zanjani E.D. Horowitz B. Sanders K.M. Purification of interstitial cells of Cajal by fluorescence-activated cell sorting.Am J Physiol Cell Physiol. 2004; 286: C448-C456Crossref PubMed Scopus (27) Google Scholar The approach to sort ICC was described previously.27Ördög T. Redelman D. Miller L.J. Horváth V.J. Zhong Q. Almeida-Porada G. Zanjani E.D. Horowitz B. Sanders K.M. Purification of interstitial cells of Cajal by fluorescence-activated cell sorting.Am J Physiol Cell Physiol. 2004; 286: C448-C456Crossref PubMed Scopus (27) Google Scholar Briefly, ICC and macrophages in the intact gastric corpus + antrum tunica muscularis were labeled with Alexa Fluor 488-ACK2 and monoclonal phycoerythrin-cyanine 5 (PC5)-anti-F4/80 (Caltag, Burlingame, CA), respectively. After the tissues were dispersed into single-cell suspensions, macrophages and dendritic cells were labeled with superparamagnetic monoclonal antibodies (Miltenyi Biotec, Auburn, CA) against CD11b and CD11c, respectively, and the labeling of macrophages was reinforced with a monoclonal PC5-anti-CD11b antibody (Caltag). Mast cells and other leukocytes were labeled with monoclonal PC5-anti-CD45 antibodies (eBioscience, San Diego, CA). Macrophages and dendritic cells were then depleted by immunomagnetic selection (Miltenyi). ICC were sorted on either a Beckman Coulter EPICS Elite or a Becton Dickinson (San Jose, CA) FACSVantage instrument. A small aliquot of the 20,000–100,000 ICC harvested in each experiment was reanalyzed by flow cytometry (FCM) on a Beckman Coulter XL/MCL for purity. Approximately 50,000 cells removed before the immunomagnetic depletion were used as unsorted control. Purified ICC, unsorted cells, and gastric muscles representing the entire anatomically defined corpus + antrum were analyzed by established techniques.26Horváth V.J. Vittal H. Ördög T. Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetes mellitus-associated depletion of interstitial cells of Cajal in the murine stomach.Diabetes. 2005; 54: 1528-1533Crossref PubMed Scopus (86) Google Scholar, 27Ördög T. Redelman D. Miller L.J. Horváth V.J. Zhong Q. Almeida-Porada G. Zanjani E.D. Horowitz B. Sanders K.M. Purification of interstitial cells of Cajal by fluorescence-activated cell sorting.Am J Physiol Cell Physiol. 2004; 286: C448-C456Crossref PubMed Scopus (27) Google Scholar, 28Ördög T. Redelman D. Horváth V.J. Miller L.J. Horowitz B. Sanders K.M. Quantitative analysis by flow cytometry of interstitial cells of Cajal, pacemakers and mediators of neurotransmission in the gastrointestinal tract.Cytometry A. 2004; 62: 139-149Crossref PubMed Scopus (27) Google Scholar The complementary DNA was amplified (40 cycles) with the following specific primers (Qiagen, Valencia, CA): β-actin (GenBank accession number: X03672 ; primers: sense bordering nucleotides: 210–231; antisense bordering nucleotides: 337–360); glyceraldehyde-3-phosphate dehydrogenase (Gapd; NM_001001303): 283–305, 440–459; c-kit (Y00864): 2706–2726, 2847–2867; Mcpt6 (M57626): 264–283, 433–454; Myh11 (NM_013607): 5721–5745, 5930–5954; Uchl1 (AF172334): 22–44, 171–190; S100b (NM_009115): 28–50, 153–177; Cd34 (NM_133654): 109–126, 280–298; Cd68 (NM_009853): 41–65, 201–225; Insr (NM_010568): 3279–3290, 3386–3405; and Igf1r (AF056187): 2208–2227, 2385–2407. Because of alternate mRNA splicing, SCF (Kitl, NM_013598) exists in 2 transmembrane forms: the longer one is subject to rapid proteolysis, which gives rise to soluble SCF. The shorter form remains membrane-bound and forms homodimers important for the activation of its receptor, c-Kit. We calculated the expression of the membrane-bound isoform in each sample by subtracting the amount of soluble SCF message (primers: 716–739, 820–843) from the amount of a transcript common to both isoforms (primers: 492–515, 639–662).28Ördög T. Redelman D. Horváth V.J. Miller L.J. Horowitz B. Sanders K.M. Quantitative analysis by flow cytometry of interstitial cells of Cajal, pacemakers and mediators of neurotransmission in the gastrointestinal tract.Cytometry A. 2004; 62: 139-149Crossref PubMed Scopus (27) Google Scholar Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was performed by using SYBR Green chemistry on a GeneAmp 5700 sequence detector (Applied Biosystems, Foster City, CA) as described.26Horváth V.J. Vittal H. Ördög T. Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetes mellitus-associated depletion of interstitial cells of Cajal in the murine stomach.Diabetes. 2005; 54: 1528-1533Crossref PubMed Scopus (86) Google Scholar, 27Ördög T. Redelman D. Miller L.J. Horváth V.J. Zhong Q. Almeida-Porada G. Zanjani E.D. Horowitz B. Sanders K.M. Purification of interstitial cells of Cajal by fluorescence-activated cell sorting.Am J Physiol Cell Physiol. 2004; 286: C448-C456Crossref PubMed Scopus (27) Google Scholar, 28Ördög T. Redelman D. Horváth V.J. Miller L.J. Horowitz B. Sanders K.M. Quantitative analysis by flow cytometry of interstitial cells of Cajal, pacemakers and mediators of neurotransmission in the gastrointestinal tract.Cytometry A. 2004; 62: 139-149Crossref PubMed Scopus (27) Google Scholar Transcriptional quantification was obtained relative to the β-actin (a housekeeping gene) standard curve and expressed in β-actin units as transcript per corpus + antrum tunica muscularis. In some experiments, Gapd was used as a reference housekeeping gene. The specificity of the primers was tested with the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/) and by sequencing the PCR products. We tested for genomic DNA contamination by PCR using cytoglobin (AJ315163) primers that span an intron (268–289, 497–519).26Horváth V.J. Vittal H. Ördög T. Reduced insulin and IGF-I signaling, not hyperglycemia, underlies the diabetes mellitus-associated depletion of interstitial cells of Cajal in the murine stomach.Diabetes. 2005; 54: 1528-1533Crossref PubMed Scopus (86) Google Scholar, 27Ördög T. Redelman D. Miller L.J. Horváth V.J. Zhong Q. Almeida-Porada G. Zanjani E.D. Horowitz B. Sanders K.M. Purification of interstitial cells of Cajal by fluorescence-activated cell sorting.Am J Physiol Cell Physiol. 2004; 286: C448-C456Crossref PubMed Scopus (27) Google Scholar, 28Ördög T. Redelman D. Horváth V.J. Miller L.J. Horowitz B. Sanders K.M. Quantitative analysis by flow cytometry of interstitial cells of Cajal, pacemakers and mediators of neurotransmission in the gastrointestinal tract.Cytometry A. 2004; 62: 139-149Cros
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