The secretory ability of newly formed secretory granules is regulated by pro-cathepsin B and amylase in parotid glands.

Parotid glands are exocrine glands that release saliva into the oral cavity. Acinar cells of parotid glands produce many secretory granules (SGs) that contain the digestion enzyme amylase. After the generation of SGs in the Golgi apparatus, they mature by enlarging and membrane remodeling. VAMP2, which is involved in exocytosis, accumulates in the membrane of mature SGs. The remodeling of SG membranes is regarded as a preparation process for exocytosis but its detailed mechanism remains unknown. To address that subject, we investigated the secretory ability of newly formed SGs. Although amylase is a useful indicator of secretion, the cell leakage of amylase might affect the measurement of secretion. Thus, in this study, we focused on cathepsin B (CTSB), a lysosomal protease, as an indicator of secretion. It has been reported that some procathepsin B (pro-CTSB), which is a precursor of CTSB, is initially sorted to SGs after which it is transported to lysosomes by clathrin-coated vesicles. Because pro-CTSB is processed to mature CTSB after its arrival in lysosomes, we can distinguish between the secretion of SGs and cell leakage by measuring the secretion of pro-CTSB and mature CTSB, respectively. When acinar cells isolated from parotid glands were stimulated with isoproterenol (Iso), a ß-adrenergic agonist, the secretion of pro-CTSB was increased. In contrast, mature CTSB was not detected in the medium although it was abundant in the cell lysates. To prepare parotid glands rich in newly formed SGs, the depletion of per-existing SGs was induced by an intraperitoneal injection of Iso into rats. At 5 h after that injection, newly formed SGs were observed in parotid acinar cells and the secretion of pro-CTSB was also detected. We confirmed that the purified newly formed SGs contained pro-CTSB, but not mature CTSB. At 2 h after Iso injection, few SGs were observed in the parotid glands and the secretion of pro-CTSB was not detected, which proved that the Iso injection depleted pre-existing SGs and the SGs observed at 5 h were newly formed after the Iso injection. These results suggest that newly formed SGs have a secretory ability prior to membrane remodeling.

Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland.

Objective: To identify factors that affect salivary gland recovery, we investigated the expression and function of bone morphogenetic protein 2 (BMP2) in mice. Materials and Methods: Using a micro clip, mice parotid glands were removed 7 days after the ligation of the unilateral parotid excretory duct. Thereafter, they were weighed and stained with hematoxylin and eosin, and BMP2 expression was examined via real-time reverse transcription-polymerase chain reaction. Primary cultures of parotid glands were prepared, and BMP2 protein was added to the culture medium for 48 hr to examine its effect on cell proliferation. E-cadherin and vimentin expression was examined using western blotting. Finally, immunohistochemical staining using an anti-Ki67 antibody was performed. Results: Duct-ligated parotid glands weighed less than those that were collected after sham surgery and showed acinar cell atrophy. They also showed higher BMP2 expression than control glands. Primary-cultured parotid acinar cells supplemented with BMP2 showed higher proliferative potential than control cells. Furthermore, they showed E-cadherin, but not vimentin, expression, and their percentage of Ki67-positive cells were higher than that corresponding to the controls. Conclusions: Injury to salivary glands by excretory duct ligation increased BMP2 expression, which may be involved in maintaining salivary gland function by inducing acinar cell proliferation.

Plasma progastrin-releasing peptide level shows different predictive profiles for treatment response by androgen receptor axis-targeted agents in patients with metastatic castration-resistant prostate cancer.

BACKGROUND: The neuroendocrine (NE) pathway cannot be ignored as a mechanism for castration-resistant prostate cancer (CRPC) progression. The neuromediator, gastrin-releasing peptide (GRP) may be involved in the aberrant activation of the normal androgen receptor (AR) and increased AR variants. This study focused on plasma levels of progastrin-releasing peptide (ProGRP) and examined the treatment outcomes with androgen receptor axis-targeted (ARAT) agents. METHODS: One hundred patients with metastatic CRPC were enrolled. Enzalutamide (ENZ) or abiraterone acetate/prednisone (AA/P) were administered to 50 patients each in a nonrandomized manner as a first-line or later choice. Plasma ProGRP levels were determined using a chemiluminescent enzyme immunoassay, and data were collected prospectively. The study endpoints were prostate-specific antigen (PSA) response and survival estimates. RESULTS: In the ENZ series, ProGRP levels correlated with the maximum PSA change from baseline (high ProGRP: -34.5% vs. low ProGRP: -85.7% p = .033). PSA progression-free survival (PFS), radiographic/symptomatic (r/s) PFS, and overall survival (OS) in patients with high ProGRP were significantly worse than those in patients with low ProGRP (median PSA-PFS: 3.3 vs. 10.0 months, p = .001, r/s PFS: 5.0 vs. 15.0 months, p < 0.001, and OS 17.5 vs. 49.0 months, p < .001, respectively). In addition, ProGRP showed an independent predictive value for all survival estimates in multivariate analyses. In the AA/P series, ProGRP levels did not correlate with the PSA change or predict PSA-PFS and r/s PFS, but they maintained a significant difference in OS (19.0 vs. 48.0 months, p = .003). CONCLUSIONS: Plasma ProGRP provides a consistent predictive value for OS in metastatic CRPC patients who underwent therapy with ARAT agents. Meanwhile, ProGRP showed different predictive profiles for PSA- and r/s PFS between ENZ and AA/P. These findings clinically suggest a mechanism for CRPC progression involving the NE pathway via the GRP. The underlying mechanism of different predictive profiles by the ARAT agent should be explored in future research.

A case of self-inserted foreign body in the urinary bladder: Usefulness of three-dimensional reconstruction computed tomography for surgery planning.

Retrieving intravesical foreign bodies warrants open cystotomy; therefore, preoperative evaluation of the material, size, shape, and location is essential for surgical planning. A 79-year-old man presented with dysuria and admitted inserting a jump rope into his urethra. Reconstructed three-dimensional computed tomography showed an entangled jump rope; therefore an endoscopic surgery was deemed unsuitable. Instead, the rope was removed through a small open cystotomy. He had no complications. Intravesical foreign bodies are not rare, and they should be considered as a differential diagnosis in patients with lower urinary tract symptoms. Three-dimensional reconstruction computed tomography contributes to surgical planning.

A case of estrogen-secreting adrenocortical carcinoma: Comprehensive immunohistochemical analysis of disorganized steroid genesis.

INTRODUCTION: Adrenocortical carcinoma rarely secretes estrogens, and little is known regarding the mechanism of intra-tumor estrogen production. We report an estrogen-secreting adrenocortical carcinoma in a postmenopausal woman, where comprehensive immunohistochemical analyses of the resected tumor revealed disorganized steroidogenesis. CASE PRESENTATION: A 68-year-old woman presented with postmenopausal vaginal bleeding and was found to have a left adrenal tumor. Serum estradiol and testosterone were elevated but they normalized after resection of the tumor, suggestive of adrenocortical carcinoma with disorganized steroidogenesis. Immunohistochemical analyses revealed that the tumor expressed aromatase which converts androgens into estrogens. Furthermore, the tumor lacked 17ßHSD2, which converts estradiol to estrone, suggesting that estradiol accumulated as the final product of the tumor's steroidogenic pathway. CONCLUSION: The capability of adrenocortical carcinoma to produce estrogen can be demonstrated by comprehensive immunohistochemical analyses of steroidogenic enzymes, such as those reported here.

Metastatic mucinous tubular and spindle cell carcinoma of the kidney responding to nivolumab plus ipilimumab.

INTRODUCTION: Mucinous tubular and spindle cell carcinoma is a rare subtype of renal cell carcinoma. Little is known regarding the efficacy of systemic therapy on its metastatic form because of its rarity. CASE PRESENTATION: We present the case of a patient with metastatic mucinous tubular and spindle cell carcinoma who achieved durable complete remission of multiple osseous metastases after undergoing cytoreductive nephrectomy followed by combination immunotherapy (ipilimumab plus nivolumab). Immunohistochemical analyses of the primary tumor revealed the presence of the tumor-infiltrating immune cells, including activated CD8+ T cells and PD-L1 expression, suggesting an immunologically hot tumor. CONCLUSION: Combination immunotherapy was a viable treatment option for this disease. Immunohistochemical analyses of the tumor-infiltrating immune cells predicted the efficacy of immune checkpoint inhibitors against rare renal cancers.

Switching of cargo sorting from the constitutive to regulated secretory pathway by the addition of cystatin D sequence in salivary acinar cells.

The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the ß-adrenergic receptor agonist isoproterenol as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, whereas fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi networks. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.NEW & NOTEWORTHY The mechanism underlying the segregation of cargo proteins to the regulated and constitutive secretory pathways in exocrine cells remains to be solved. We analyzed unstimulated secretion in salivary acinar cells by performing double-labeling experiments using HaloTag technology and computer simulation. It revealed that the majority of HaloTag with only signal peptide sequence was secreted through the constitutive pathway and that the addition of a full-length cystatin D sequence changed its sorting to the regulated pathway.

Suppression of parotid acinar cell dysfunction by the free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one.

Salivary gland atrophy and consequent hyposalivation are serious problems in clinical dentistry, as saliva regulates the environment of the oral cavity. To clarify the mechanisms underlying salivary gland dysfunction, a system for primary culture of parotid acinar cells has been established. It has been reported previously that the process of cell isolation from parotid glands triggers stress signaling mediated by Src and p38 mitogen-activated protein (MAP) kinase (p38), leading to dedifferentiation of acinar cells, and that an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor suppresses this activation of Src and p38, suggesting that reactive oxygen species initiate the dedifferentiation signal. The present study examined the effect of a free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (also termed MCI-186 or edaravone), on activation of the stress signal and the secretory function of parotid acinar cells. Activation of p38 during cell isolation was suppressed by addition of MCI-186. The retention of the activity of amylase, a major salivary protein, and the number of amylase-containing secretory granules were improved by isolation and culture in the presence of MCI-186. In addition, calcium elevation upon stimulation with a muscarinic agonist was higher in MCI-186-treated cells than in untreated cells. These results suggest that MCI-186 (edaravone) is a promising agent for prevention of salivary gland dysfunction.

Maintenance of claudin-3 expression and the barrier functions of intercellular junctions in parotid acinar cells via the inhibition of Src signaling.

OBJECTIVES: Salivary acinar and duct cells show different expression patterns of claudins, which may reflect their different functions. To study the role of claudins in saliva secretion, we examined alterations in the expression patterns of cell adhesion molecules in parotid glands of γ-irradiated rats and analyzed the influence of those changes on intercellular barrier function using primary cultures of parotid acinar cells. DESIGN: Rats were γ-irradiated with doses of 5, 15 or 20Gy, and expression levels of cell adhesion molecules were examined by immunoblotting analysis. Acinar cells were isolated from parotid glands and were cultured in the absence or presence of the Src kinase inhibitor PP1. Changes in protein and mRNA expression patterns were determined by immunoblotting and by RT-PCR analyses, respectively. Intercellular barrier function was examined by measuring transepithelial electrical resistance and the paracellular flux of FITC-dextran. RESULTS: In irradiated parotid glands, the expression of claudin-4 was enhanced at 15Gy or higher, levels that induce the hyposecretion of saliva, although that increase was transient. At 30days after irradiation, expression levels of cell adhesion molecules were decreased. In primary cultures, the expression of claudin-4 was also increased transiently but the expression of claudin-3 and E-cadherin was decreased. The barrier function of tight junctions was disrupted although the localization of occludin was maintained. The Src kinase inhibitor PP1 suppressed those changes in gene expression and retained the intercellular barrier function. CONCLUSIONS: These results suggest that the inhibition of Src signaling maintains the barrier functions of intercellular junctions in salivary glands, which can be lost due to tissue injury.

Secretory proteins without a transport signal are retained in secretory granules during maturation in rat parotid acinar cells.

OBJECTIVE: The acinar cells of the parotid gland are filled with numerous secretory granules (SGs), which accumulate the digestion enzyme amylase. SGs mature accompanied with membrane remodelling such as fusion and budding of small vesicles. However, little is understood about the mechanism of the condensation of SG contents during maturation. In this study, we examined whether secretory proteins need a specific signal to be retained in SGs. DESIGN: To induce internalization of the luminal membrane after exocytosis, we injected the ß-adrenergic agonist isoproterenol into rats. Acinar cells were then incubated with Lucifer Yellow (LY) dye as a tracer for 3h for uptake into immature secretory granules (ISGs). To observe whether LY was retained in SGs after maturation, we continued incubating the cultured acinar cells for 2 days. RESULTS: The localization of LY into ISGs was confirmed by the following four methods: (1) co-localization of the fluorescence of LY and amylase by confocal laser microscopy, (2) detection of the fluorescence from purified ISGs, (3) secretion of the fluorescence together with amylase upon stimulation, and (4) observation of the intracellular localization of LY by electron microscopy. Moreover, we observed co-localization of some of the SGs with the fluorescence of LY after cell culture. CONCLUSIONS: Although the fusion and budding of small vesicles may contribute to the process of granule maturation, LY remained in the SGs even after maturation. These results suggest that secretory proteins that have no transport signal are not excluded from SGs, and they are retained in SGs during granule maturation in exocrine parotid glands.

Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.

The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a ß-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.