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1.
Int J Mol Sci ; 22(22)2021 Nov 18.
Article in English | MEDLINE | ID: mdl-34830342

ABSTRACT

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.


Subject(s)
Escherichia coli Infections/genetics , Osteopontin/genetics , Prostate/metabolism , Urinary Tract Infections/genetics , Uropathogenic Escherichia coli/pathogenicity , Animals , Chondroitin Sulfate Proteoglycans/genetics , Chondroitin Sulfate Proteoglycans/metabolism , Collagen Type III/genetics , Collagen Type III/metabolism , Disease Models, Animal , Escherichia coli Infections/metabolism , Escherichia coli Infections/pathology , Escherichia coli Infections/prevention & control , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Fibrosis , Gene Expression Regulation , Humans , Inflammation , Lumican/genetics , Lumican/metabolism , Male , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteopontin/deficiency , Prostate/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Urinary Tract Infections/metabolism , Urinary Tract Infections/pathology , Urinary Tract Infections/prevention & control , Uropathogenic Escherichia coli/growth & development
2.
Am J Physiol Renal Physiol ; 320(1): F31-F46, 2021 01 01.
Article in English | MEDLINE | ID: mdl-33135480

ABSTRACT

Bacterial infection is one known etiology of prostatic inflammation. Prostatic inflammation is associated with prostatic collagen accumulation and both are linked to progressive lower urinary tract symptoms in men. We characterized a model of prostatic inflammation using transurethral instillations of Escherichia coli UTI89 in C57BL/6J male mice with the goal of determining the optimal instillation conditions, understanding the impact of instillation conditions on urinary physiology, and identifying ideal prostatic lobes and collagen 1a1 prostatic cell types for further analysis. The smallest instillation volume tested (50 µL) distributed exclusively to the bladder, 100- and 200-µL volumes distributed to the bladder and prostate, and a 500-µL volume distributed to the bladder, prostate, and ureter. A threshold optical density of 0.4 E. coli UTI89 in the instillation fluid was necessary for significant (P < 0.05) prostate colonization. E. coli UTI89 infection resulted in a low frequency, high volume spontaneous voiding pattern. This phenotype was due to exposure to E. coli UTI89, not catheterization alone, and was minimally altered by a 50-µL increase in instillation volume and doubling of E. coli concentration. Prostate inflammation was isolated to the dorsal prostate and was accompanied by increased collagen density. This was partnered with increased density of protein tyrosine phosphatase receptor type C+, procollagen type I-α1+ copositive cells and decreased density of α2-smooth muscle actin+, procollagen type I-α1+ copositive cells. Overall, we determined that this model is effective in altering urinary phenotype and producing prostatic inflammation and collagen accumulation in mice.


Subject(s)
Collagen Type I/metabolism , Escherichia coli Infections/microbiology , Procollagen/metabolism , Prostate/microbiology , Prostatitis/microbiology , Uropathogenic Escherichia coli/pathogenicity , Actins/metabolism , Animals , Collagen Type I, alpha 1 Chain , Disease Models, Animal , Escherichia coli Infections/complications , Leukocyte Common Antigens/metabolism , Male , Mice, Inbred C57BL , Prostate/metabolism , Prostate/pathology , Prostatitis/metabolism , Prostatitis/pathology , Tissue Culture Techniques
3.
Prostate ; 80(11): 872-884, 2020 08.
Article in English | MEDLINE | ID: mdl-32497356

ABSTRACT

BACKGROUND: Castration-insensitive epithelial progenitors capable of regenerating the prostate have been proposed to be concentrated in the proximal region based on facultative assays. Functional characterization of prostate epithelial populations isolated with individual cell surface markers has failed to provide a consensus on the anatomical and transcriptional identity of proximal prostate progenitors. METHODS: Here, we use single-cell RNA sequencing to obtain a complete transcriptomic profile of all epithelial cells in the mouse prostate and urethra to objectively identify cellular subtypes. Pan-transcriptomic comparison to human prostate cell types identified a mouse equivalent of human urethral luminal cells, which highly expressed putative prostate progenitor markers. Validation of the urethral luminal cell cluster was performed using immunostaining and flow cytometry. RESULTS: Our data reveal that previously identified facultative progenitors marked by Trop2, Sca-1, KRT4, and PSCA are actually luminal epithelial cells of the urethra that extend into the proximal region of the prostate, and are resistant to castration-induced androgen deprivation. Mouse urethral luminal cells were identified to be the equivalent of previously identified human club and hillock cells that similarly extend into proximal prostate ducts. Benign prostatic hyperplasia (BPH) has long been considered an "embryonic reawakening," but the cellular origin of the hyperplastic growth concentrated in the periurethral region is unclear. We demonstrate an increase in urethral luminal cells within glandular nodules from BPH patients. Urethral luminal cells are further increased in patients treated with a 5-α reductase inhibitor. CONCLUSIONS: Our data demonstrate that cells of the proximal prostate that express putative progenitor markers, and are enriched by castration in the proximal prostate, are urethral luminal cells and that these cells may play an important role in the etiology of human BPH.


Subject(s)
Prostate/cytology , Stem Cells/cytology , Urethra/cytology , Adolescent , Adult , Animals , Antigens, Neoplasm/metabolism , Cell Adhesion Molecules/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Prostate/metabolism , Stem Cells/metabolism , Urethra/metabolism , Young Adult
4.
Histochem Cell Biol ; 152(1): 35-45, 2019 Jul.
Article in English | MEDLINE | ID: mdl-30976911

ABSTRACT

Prostate autonomic and sensory axons control glandular growth, fluid secretion, and smooth muscle contraction and are remodeled during cancer and inflammation. Morphogenetic signaling pathways reawakened during disease progression may drive this axon remodeling. These pathways are linked to proliferative activities in prostate cancer and benign prostate hyperplasia. However, little is known about which developmental signaling pathways guide axon investment into prostate. The first step in defining these pathways is pinpointing when axon subtypes first appear in prostate. We accomplished this by immunohistochemically mapping three axon subtypes (noradrenergic, cholinergic, and peptidergic) during fetal, neonatal, and adult stages of mouse prostate development. We devised a method for peri-prostatic axon density quantification and tested whether innervation is uniform across the proximo-distal axis of dorsal and ventral adult mouse prostate. Many axons directly interact with or innervate neuroendocrine cells in other organs, so we examined whether sensory or autonomic axons innervate neuroendocrine cells in prostate. We first detected noradrenergic, cholinergic, and peptidergic axons in prostate at embryonic day (E) 14.5. Noradrenergic and cholinergic axon densities are uniform across the proximal-distal axis of adult mouse prostate while peptidergic axons are denser in the periurethral and proximal regions. Peptidergic and cholinergic axons are closely associated with prostate neuroendocrine cells whereas noradrenergic axons are not. These results provide a foundation for understanding mouse prostatic axon development and organization and, provide strategies for quantifying axons during progression of prostate disease.


Subject(s)
Axons/metabolism , Prostate/embryology , Prostate/innervation , Animals , Axons/pathology , Male , Mice , Mice, Inbred C57BL , Prostate/cytology , Prostate/pathology
5.
PLoS One ; 12(11): e0188413, 2017.
Article in English | MEDLINE | ID: mdl-29145476

ABSTRACT

Though many methods can be used to identify cell types contained in complex tissues, most require cell disaggregation and destroy information about where cells reside in relation to their microenvironment. Here, we describe a polytomous key for cell type identification in intact sections of adult mouse prostate and prostatic urethra. The key is organized as a decision tree and initiates with one round of immunostaining for nerve, epithelial, fibromuscular/hematolymphoid, or vascular associated cells. Cell identities are recursively eliminated by subsequent staining events until the remaining pool of potential cell types can be distinguished by direct comparison to other cells. We validated our identification key using wild type adult mouse prostate and urethra tissue sections and it currently resolves sixteen distinct cell populations which include three nerve fiber types as well as four epithelial, five fibromuscular/hematolymphoid, one nerve-associated, and three vascular-associated cell types. We demonstrate two uses of this novel identification methodology. We first used the identification key to characterize prostate stromal cell type changes in response to constitutive phosphatidylinositide-3-kinase activation in prostate epithelium. We then used the key to map cell lineages in a new reporter mouse strain driven by Wnt10aem1(cre/ERT2)Amc. The identification key facilitates rigorous and reproducible cell identification in prostate tissue sections and can be expanded to resolve additional cell types as new antibodies and other resources become available.


Subject(s)
Prostate/metabolism , Urethra/metabolism , Animals , Immunohistochemistry , Male , Mice , Prostate/cytology , Urethra/cytology
6.
Endocrinology ; 158(6): 1645-1658, 2017 06 01.
Article in English | MEDLINE | ID: mdl-28419211

ABSTRACT

The α-subunit of the heterotrimeric Gz protein, Gαz, promotes ß-cell death and inhibits ß-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gαz protein would preserve functional ß-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in Gαz knockout mice. By 17 weeks of age, Gαz-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive ß-cells in Gαz-null islets despite similar immune infiltration in control mice. Islets from Gαz-null mice also exhibited a higher percentage of Ki-67-positive ß-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, ß-cell-specific Gαz-null mice phenocopy whole-body Gαz-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a ß-cell-centric role for Gαz in diabetes pathophysiology. We propose that Gαz plays a key role in ß-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of ß-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response.


Subject(s)
Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Type 1/genetics , GTP-Binding Protein alpha Subunits/genetics , Animals , Apoptosis/genetics , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 1/pathology , Female , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, Transgenic , Streptozocin
7.
Mol Endocrinol ; 30(5): 543-56, 2016 May.
Article in English | MEDLINE | ID: mdl-27049466

ABSTRACT

A defining characteristic of type 1 diabetes mellitus (T1DM) pathophysiology is pancreatic ß-cell death and dysfunction, resulting in insufficient insulin secretion to properly control blood glucose levels. Treatments that promote ß-cell replication and survival, thus reversing the loss of ß-cell mass, while also preserving ß-cell function, could lead to a real cure for T1DM. The α-subunit of the heterotrimeric Gz protein, Gαz, is a tonic negative regulator of adenylate cyclase and downstream cAMP production. cAMP is one of a few identified signaling molecules that can simultaneously have a positive impact on pancreatic islet ß-cell proliferation, survival, and function. The purpose of our study was to determine whether mice lacking Gαz might be protected, at least partially, from ß-cell loss and dysfunction after streptozotocin treatment. We also aimed to determine whether Gαz might act in concert with an activator of the cAMP-stimulatory glucagon-like peptide 1 receptor, exendin-4 (Ex4). Without Ex4 treatment, Gαz-null mice still developed hyperglycemia, albeit delayed. The same finding held true for wild-type mice treated with Ex4. With Ex4 treatment, Gαz-null mice were protected from developing severe hyperglycemia. Immunohistological studies performed on pancreas sections and in vitro apoptosis, cytotoxicity, and survival assays demonstrated a clear effect of Gαz signaling on pancreatic ß-cell replication and death; ß-cell function was also improved in Gαz-null islets. These data support our hypothesis that a combination of therapies targeting both stimulatory and inhibitory pathways will be more effective than either alone at protecting, preserving, and possibly regenerating ß-cell mass and function in T1DM.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , Glucagon-Like Peptide 1/metabolism , Insulin-Secreting Cells/metabolism , Adenylyl Cyclases/metabolism , Animals , Blood Glucose/drug effects , Blood Glucose/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/physiology , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Type 1/chemically induced , Diabetes Mellitus, Type 1/metabolism , Exenatide , Glucose/metabolism , Hyperglycemia/drug therapy , Hyperglycemia/metabolism , Hypoglycemic Agents/pharmacology , Insulin/metabolism , Insulin-Secreting Cells/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Pancreas/drug effects , Pancreas/metabolism , Peptides/metabolism , Rats , Signal Transduction/drug effects , Streptozocin/pharmacology , Venoms/metabolism
8.
Am J Physiol Gastrointest Liver Physiol ; 309(6): G431-42, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26185331

ABSTRACT

Stimulation of digestive organs by enteric peptides is lost during total parental nutrition (PN). Here we examine the role of the enteric peptide bombesin (BBS) in stimulation of the exocrine and endocrine pancreas during PN. BBS protects against exocrine pancreas atrophy and dysfunction caused by PN. BBS also augments circulating insulin levels, suggesting an endocrine pancreas phenotype. While no significant changes in gross endocrine pancreas morphology were observed, pancreatic islets isolated from BBS-treated PN mice showed a significantly enhanced insulin secretion response to the glucagon-like peptide-1 (GLP-1) agonist exendin-4, correlating with enhanced GLP-1 receptor expression. BBS itself had no effect on islet function, as reflected in low expression of BBS receptors in islet samples. Intestinal BBS receptor expression was enhanced in PN with BBS, and circulating active GLP-1 levels were significantly enhanced in BBS-treated PN mice. We hypothesized that BBS preserved islet function indirectly, through the enteroendocrine cell-pancreas axis. We confirmed the ability of BBS to directly stimulate intestinal enteroid cells to express the GLP-1 precursor preproglucagon. In conclusion, BBS preserves the exocrine and endocrine pancreas functions during PN; however, the endocrine stimulation is likely indirect, through the enteroendocrine cell-pancreas axis.


Subject(s)
Bombesin/pharmacology , Gastrin-Releasing Peptide/analogs & derivatives , Islets of Langerhans/drug effects , Pancreas, Exocrine/drug effects , Parenteral Nutrition/adverse effects , Amylases/metabolism , Animals , DNA/metabolism , Food, Formulated , Gene Expression Regulation , Hyperglycemia/blood , Islets of Langerhans/anatomy & histology , Lipase/metabolism , Male , Mice , Mice, Inbred ICR , Pancreas, Exocrine/anatomy & histology , Pancreatic Hormones/metabolism
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