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High-risk pregnancies, such as pregnancies with gestational diabetes mellitus (GDM), are becoming more common and as such, have become important public health issues worldwide. GDM increases the risks of macrosomia, premature infants, and preeclampsia. Although placental dysfunction, including fibrosis is associated with the development of GDM, factors that link these observations remain unknown. Prothymosin α (ProTα) is expressed in the placenta and is involved in cell proliferation and immunomodulation. It also plays an important role in insulin resistance and fibrosis. However, the role of ProTα in GDM is still unclear. In the present study, we found that fibrosis-related protein expressions, such as type I collagen (Col-1) were significantly increased in the placentae of ProTα transgenic mice. With elevated fibrosis-related protein expressions, placental weights significantly increased in GDM group. In addition, placental and circulating ProTα levels were significantly higher in patients with GDM (n=39), compared with the healthy group (n=102), and were positively correlated with Col-1 expression. Mice with streptozotocin (STZ)-induced GDM had increased ProTα, fasting blood glucose, Col-1, and placental weight, whereas plasma insulin levels were decreased. ProTα overexpression enhanced nuclear factor κB (NFκB) activation to increase fibrosis-related protein expressions in 3A-Sub-E trophoblasts, while treatment with an NFκB inhibitor reversed the effect of ProTα on fibrosis-related protein expressions. We further investigated whether ProTα is regulated by hyperglycemia-induced reactive oxygen species (ROS). In conclusion, ProTα increases the amount of placental connective tissue and thus contributes to the pathogenesis of placental fibrosis in GDM. Therefore, ProTα may be a novel therapeutic target for GDM., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Polycystic kidney disease (PKD) is characterized by the expansion of fluid-filled cysts in the kidney, which impair the function of kidney and eventually leads to end-stage renal failure. It has been previously demonstrated that transgenic overexpression of prothymosin α (ProT) induces the development of PKD; however, the underlying mechanisms remain unclear. In this study, we used a mouse PKD model that sustains kidney-specific low-expression of Pkd1 to illustrate that aberrant up-regulation of ProT occurs in cyst-lining epithelial cells, and we further developed an in vitro cystogenesis model to demonstrate that the suppression of ProT is sufficient to reduce cyst formation. Next, we found that the expression of ProT was accompanied with prominent augmentation of protein acetylation in PKD, which results in the activation of downstream signal transducer and activator of transcription (STAT) 3. The pathologic role of STAT3 in PKD has been previously reported. We determined that this molecular mechanism of protein acetylation is involved with the interaction between ProT and STAT3; consequently, it causes the deprivation of histone deacetylase 3 from the indicated protein. Conclusively, these results elucidate the significant role of ProT, including protein acetylation and STAT3 activation in PKD, which represent potential for ameliorating the disease progression of PKD.-Chen, Y.-C., Su, Y.-C., Shieh, G.-S., Su, B.-H., Su, W.-C., Huang, P.-H., Jiang, S.-T., Shiau, A.-L., Wu, C.-L. Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Prothymosin-α (PTMA) is a small, acidic protein that is usually transported into the nucleus and involves many cellular and immunological functions. Previous studies demonstrated that aberrant location of PTMA expression exists in human bladder cancer, but the role of PTMA protein expression remains elusive. In this study, we created ectopic nuclear or cytoplasmic PTMA expression in human bladder cancer cells by infecting lentiviruses carrying wild type or deleted nuclear localization signal of the PTMA gene. The in vivo tumorigenesis assay showed PTMA protein with deleted nuclear localization signal promotes J82 xenograft tumor growth in mice and shortens their survival more so than the wild type. Chromatin immunoprecipitation showed that wild-type PTMA protein binds to the PTEN promoter and enhances phosphatase and tensin homolog (PTEN) expression. Through immunoblot proteomics and in vivo ubiquitination studies, PTMA protein can bind with tripartite motif-containing protein 21 (TRIM21) and block its ubiquitination. Also, TRIM21 can downregulate both forms of PTMA protein. In human bladder tumors, loss of nuclear PTMA expression was an unfavorable prognostic indicator for shorter disease-free survival (hazard ratio, 1.54; P = 0.009). Our data support that nuclear PTMA protein serves as a tumor suppressor in bladder cancer through upregulating PTEN and orchestrating TRIM21 for the regulation of Nrf2 signaling., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Post-ischemic inflammation plays an important role in the progression of ischemia/reperfusion injuries. Prothymosin-α (ProT) can protect cells from necrotic death following ischemia; however, its immunostimulatory actions may counteract the neuroprotective effect. We proposed that ProTΔNLS, synthesized by deleting its nuclear localizing signal (NLS) at the C-terminal of ProT, can attenuate the immunostimulatory activity and has more salient neuroprotective effect. In this study, we examined the therapeutic effects of ProT and ProTΔNLS in a transient middle cerebral artery occlusion (tMCAO) model of rats. Rats that had sustained 90 min of tMCAO were treated with GST-vehicle, ProT, or ProTΔNLS. Therapeutic outcomes were evaluated by infarction volume assay and behavioral assessment. Changes to inflammatory mediators, including tumor necrosis factor α (TNF-α), interleukin-10 (IL-10), and myeloperoxidase (MPO) were evaluated by enzyme-linked immunosorbent assay. Activated matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) levels were evaluated by gelatin zymography. Microglial activation was identified by double-immunostaining for Iba-1 and CD68. Our results showed that while both ProT and ProTΔNLS reduce infarction volume and improve functional outcome, ProTΔNLS provides the best therapeutic outcome. ProT increases TNF-α but decreases IL-10 secretion after ischemic injury, reflecting its pro-inflammatory activity. ProTΔNLS suppresses expression of TNF-α, MPO, and activity of MMPs in ischemic brain tissue. It also suppresses activation of microglia in penumbral cortex. These data demonstrate the immunesuppressive activities of ProTΔNLS. In conclusion, ProT has pro-inflammatory effect that may counteract its neuroprotective effect. Deletion of NLS from ProT may attenuate post-ischemic inflammation and enhance the neuroprotective effects of ProT.

Emphysema, a major consequence of chronic obstructive pulmonary disease (COPD), is characterized by the permanent airflow restriction resulting from enlargement of alveolar airspace and loss of lung elasticity. Transforming growth factor-β (TGFβ) signalling regulates the balance of matrix metalloproteinase (MMP)/tissue inhibitor of matrix metalloproteinase (TIMP) to control matrix homeostasis. Patients with COPD have dysregulated TGFβ signalling and reduced histone deacetylase (HDAC) activity through epigenetic up-regulation of histone acetylation in the promoters of pro-inflammatory genes. However, the potential link between decreased HDAC activity and dysregulated TGFβ signalling in emphysema pathogenesis remains to be determined. Prothymosin α (ProT), a highly conserved acidic nuclear protein, plays a role in the acetylation of histone and non-histone proteins. The aim of this study was to test the hypothesis that ProT inhibits TGFβ-Smad signalling through Smad7, thereby contributing to emphysema pathogenesis. We show that ProT enhances Smad7 acetylation by decreasing its association with HDAC and thereby down-regulates TGFβ-Smad signalling. ProT caused an imbalance between MMP and TIMP through acetylated Smad7 in favour of MMP expression. In addition to interfering with R-Smad activation and targeting receptors for degradation in the cytoplasm, acetylated Smad7 potentiated by ProT competitively antagonized binding of the pSmad2/3-Smad4 complex to the TIMP-3 promoter, resulting in reduced TIMP-3 expression. These effects were detected in ProT-over-expressing cells, lungs of ProT transgenic mice displaying an emphysema phenotype and in emphysema patients. Importantly, increased Smad7 and reduced TIMP-3 were found in the lungs of emphysema patients and mice with cigarette smoke extract (CSE)-induced emphysema. Such effects could be abrogated by silencing endogenous ProT expression. Collectively, our results uncover acetylated Smad7 regulated by ProT as an important determinant in dysregulated TGFβ signalling that contributes to emphysema pathogenesis., (Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Context: Prothymosin-α (ProT) is involved in oxidative stress, inflammation, cell proliferation, and apoptosis. Increased oxidative stress and chronic inflammation participate in the pathogenesis of diabetes. A recent study found that ProT is a ligand of toll-like receptor 4, which plays an important role in the development of insulin resistance. However, its physiological role remains poorly understood., Objective: The objective was to investigate whether ProT contributes to the development of insulin resistance., Design, Settings, and Patients: A total of 185 subjects were recruited and classified into nondiabetes (n = 95) and newly diagnosed diabetes (n = 90) groups. Transgenic mice overexpressing ProT were used to investigate the role of ProT in the development of insulin resistance. Lentiviral vectors carrying short hairpin RNA specific for ProT were delivered via the portal vein to silence hepatic ProT expression in mice with high-fat diet-induced insulin resistance. Glucose uptake was determined in L6 myotubes., Results: We show that the serum ProT levels of patients with type 2 diabetes were significantly higher than those of normal individuals (mean ± SEM, 419.8 ± 46.47 vs 246.4 ± 27.89 pg/mL; P < .001). Furthermore, ProT transgenic mice exhibited an insulin-resistant phenotype, whereas the silencing of hepatic ProT expression ameliorated high-fat diet-induced insulin resistance in C57BL/6 mice. In vitro studies reveal that ProT induced insulin resistance through a toll-like receptor 4-nuclear factor-κB-dependent pathway., Conclusions: Our results support the role for ProT in the development of insulin resistance. Therefore, ProT is a potential novel therapeutic target for type 2 diabetes.

In this paper, we report a study on the clinical relevance of prothymosin-α expression and its correlation with intratumoral Foxp3(+) and CD8(+) lymphocytes (Foxp3(+)TIL and CD8(+)TIL) in bladder cancer patients. We used immunohistochemical staining for prothymosin-α, Foxp3, and CD8 on 101 tumor specimens harvested by endoscopic resection. The results were correlated with clinicopathological variables and clinical outcome in bladder cancer patients, particularly in 73 patients with superficial disease, using the log-rank test and Cox proportional hazard model. Overall, of the tumors, 30 % were negative, 34 % showed nuclear, and 37 % showed cytoplasmic prothymosin-α expression. Foxp3(+)TILs were detected in 11 % of patients (nonnuclear vs. nuclear, p = 0.096). Patients with a history of urothelial carcinoma have a higher frequency of nonnuclear prothymosin-α expression than those without (p = 0.016, chi-square test). By univariate and multivariate analyses of cases with superficial disease, grade and stage were identified as independent predictors for recurrence-free survival (p = 0.016 and 0.016, respectively). Higher stage and nonnuclear prothymosin-α expression independently predict shorter progression-free survival (p = 0.006 and 0.043, respectively). The presence of Foxp3(+)TILs was significantly associated with disease progression by univariate analysis (p = 0.022), but not by multivariate analysis (p = 0.147). In vitro assays showed that J82 cells which express ectopically nuclear prothymosin-α exhibit higher growth rate and secrete less TGF-β1 than those with cytoplasmic expression or control cells. Altogether, prothymosin-α expression is a determinant of disease progression in superficial bladder cancer. Foxp3(+)TILs tend to be found more often in bladder cancer with nonnuclear prothymosin-α expression. Future study is required to unravel their interaction.

Prothymosin α (ProT) is involved in regulating expression of the oxidative stress-protective genes and it also exerts immunomodulatory activities. In this study, we investigated the therapeutic effects of ProT gene transfer on atherosclerosis in endothelial cells and in ApoE-deficient mice. Adenoviruses encoding mouse ProT (AdProT) were used for the management of atherosclerosis. In vitro, the effects of ProT on antioxidant gene expressions and the protection effect against oxidant-mediated injury in endothelial cells were examined. In vivo, AdProT were administered intraventricularly into the heart of ApoE(-/-) mice. Histopathological and immunohistochemical assessments of the aortic tissues were performed. Expressions of HO-1 and antioxidant genes in the aortic tissues were also determined. Our results demonstrated that ProT gene transfer increased antioxidant gene expressions, eNOS expression and NO release, as well as reduced the reactive oxygen species production in endothelial cells. Intraventricular administration of AdProT reduced the lesion formation, increased expressions of HO-1 and SOD genes, and reduced infiltrating macrophages in the aorta of ApoE(-/-) mice. This study suggests that ProT gene transfer may have the therapeutic potential for the management of atherosclerosis via inducing antioxidant gene expressions, eNOS expression and NO release, reducing ROS production and macrophage infiltration in endothelium.

Emphysema is one of the disease conditions that comprise chronic obstructive pulmonary disease. Prothymosin α transgenic mice exhibit an emphysema phenotype, but the pathophysiological role of prothymosin α in emphysema remains unclear. Here we show that prothymosin α contributes to the pathogenesis of emphysema by increasing acetylation of histones and nuclear factor-kappaB, particularly upon cigarette smoke exposure. We find a positive correlation between prothymosin α levels and the severity of emphysema in prothymosin α transgenic mice and emphysema patients. Prothymosin α overexpression increases susceptibility to cigarette smoke-induced emphysema, and cigarette smoke exposure further enhances prothymosin α expression. We show that prothymosin α inhibits the association of histone deacetylases with histones and nuclear factor-kappaB, and that prothymosin α overexpression increases expression of nuclear factor-kappaB-dependent matrix metalloproteinase 2 and matrix metalloproteinase 9, which are found in the lungs of patients with chronic obstructive pulmonary disease. These results demonstrate the clinical relevance of prothymosin α in regulating acetylation events during the pathogenesis of emphysema.

Objectives: To investigate the prognostic role of prothymosin-alpha (PTMA) expression in human upper urinary tract transitional cell carcinoma (UUT-TCC)., Methods: Paraffin-embedded tissues were collected from 91 patients with UUT-TCC and from 15 paired normal renal cortex and 13 paired urothelial walls. The primary antibody for PTMA (2F11) used was validated in 4 human urothelial cancer cell lines before assessing the surgical specimen. Immunohistochemistry was then conducted to determine the expression intensity of PTMA, the calculation of immunostaining density using imaging analysis, and for immunostaining localization. The correlates with clinicopathologic characteristics and patient survival were explored., Results: The expression intensity of PTMA demonstrated a significant enhancement of PTMA expression in UUT-TCCs compared with both paired normal tissues (P = .0002 and P = .0004 for UUT-TCC vs the urothelial wall and vs the renal cortex, respectively). As for the localization of PTMA immunoreactivity, of the 91 tumor specimens, 33 (36.3%) were cytoplasmic PTMA-expressing, 51 (56.0%) were nuclear PTMA-expressing, and 7 (7.7%) were PTMA-negative tumors. On univariate and multivariate analyses, PTMA expression localization was the sole independent prognostic indicator for recurrence-free survival (hazard ratio 4.90, 95% confidence interval 1.73-13.9; P = .003), although pathologic staging was an independent prognostic indicator for both progression-free survival (hazard ratio 22.6, 95% confidence interval 2.56-198; P = .005) and disease-specific overall survival (hazard ratio 5.60, 95% confidence interval 1.48-21.2; P = .011). The limitations of our study included small patient numbers and short follow-up., Conclusions: The results of our study have shown that PTMA is overexpressed in UUT-TCCs and that cytoplasmic PTMA expression can provide significant prognostic information for subsequent tumor recurrence in the residual urinary tract after nephroureterectomy.

Objectives: To investigate the prothymosin-alpha (PTMA) expression in human bladder cancer using tissue microarrays., Methods: Two tissue microarray slides of 50 bladder tumors and 42 paired normal adjacent tissues were investigated using immunohistochemical staining. The staining distribution was categorized as negative, nuclear, cytoplasmic, and mixed expression. Quantitative immunoreactivity was measured using image analysis, as represented by the integrated optical density for each tissue core., Results: In 36 of 42 normal adjacent tissues, positive PTMA immunoreactivity could be seen in some nuclei of the normal urothelial cells, but not, or only minimally, in the cytoplasm and underlying submucosal tissues. A statistically significant enhancement of PTMA expression was found in bladder tumors of each grade compared with the normal adjacent tissue (P < .0001 for normal adjacent tissues vs grade 1, 2, or 3 tumors, paired t test). Of 48 transitional cell carcinoma specimens, only 4 (8.3%) were graded as negative and 44 (91.7%) were positive for PTMA expression, including nuclear (n = 8), cytoplasmic (n = 12), and mixed expression (n = 24) patterns. A statistically significant correlation was found between high grade and mixed expression (P = 0.0020, chi(2) test)., Conclusions: Increased PTMA expression was found in human bladder cancers compared with the paired normal adjacent bladder tissue. The distribution of PTMA expression was changed in high-grade tumors. The clinical significance of such an aberrant PTMA expression in bladder cancer is worthy of additional investigation.

Prothymosin alpha (ProT) is regulated by c-Myc, an oncoprotein overexpressed in synovium of rheumatoid arthritis, and is associated with cell proliferation. However, ProT also exerts immunomodulatory activities. The growth-promoting activity of ProT can be abolished by deleting its nuclear localization signal (NLS). In this study, we showed that AdProTDeltaNLS, an adenoviral vector encoding ProT lacking the NLS, did not enhance the proliferation of synovial fibroblasts. AdProTDeltaNLS treatment abolished the up-regulation of the MIP-1alpha promoter activity induced by TNF-alpha in synovial fibroblasts. AdProTDeltaNLS suppressed macrophage chemotaxis and reduced macrophage infiltration into the ankle joints in rats with collagen-induced arthritis (CIA). Neutralization test confirmed the involvement of MIP-1alpha in macrophage chemotaxis. Administration of AdProTDeltaNLS reduced the severity of CIA in the clinical, radiographic, and histological aspects. The levels of TNF-alpha (mean +/- SEM, 1261.9 +/- 107.9 vs 2880.1 +/- 561.4 pg/mg total protein; p < 0.05), IL-1beta (56.8 +/- 8.0 vs 109.2 +/- 4.9 pg/mg total protein; p < 0.01), and MIP-1alpha (41.7 +/- 3.6 vs 55.2 +/- 1.1 pg/mg total protein; p < 0.05) in the ankle joints were lower in the AdProTDeltaNLS-treated rats with CIA than those in their control counterparts. In the AdProTDeltaNLS-treated ankle joints, matrix metalloproteinase-9 expression was decreased by 40% and infiltrating macrophages reduced by 50%. Our results demonstrate that intra-articular delivery of AdProTDeltaNLS significantly ameliorated the clinical course of CIA in rats. This study is the first to suggest that ProT lacking the NLS may have therapeutic potential for the management of rheumatoid arthritis.

Objectives: To establish the normal range of urine prothymosin-alpha in humans and to investigate its role as a specific tumor marker for the detection and follow-up of transitional cell carcinoma of the urinary bladder before and after curative treatment., Methods: Urine samples were obtained from 151 healthy volunteers, 60 patients with urinary tract infection, 238 patients with bladder transitional cell carcinoma (96 with tumor and 142 tumor free), and 22 patients with non-transitional cell carcinoma tumors. The urine prothymosin-alpha levels were quantified by enzyme-linked immunosorbent assay and then appropriately analyzed and compared among the different study groups., Results: The mean value of urine prothymosin-alpha in healthy volunteers was 0.68 +/- 0.13 ng/mL. Regardless of the presence of urinary tract infection, the urine prothymosin-alpha level in patients with newly diagnosed, as yet untreated, bladder cancer was significantly greater than that in those who were tumor free after curative treatment (P = 0.050 and P = 0.026 for the presence and absence of urinary tract infection, respectively). At follow-up, the urine prothymosin-alpha level was constantly elevated when residual or recurrent tumor was present after treatment. Although the urine prothymosin-alpha level in patients with non-transitional cell carcinoma tumors was not significantly different from that of healthy volunteers, it was definitely lower than the level in patients with bladder tumors (P = 0.003)., Conclusions: Our findings have revealed that urine prothymosin-alpha has the potential of being a useful tumor marker for the detection and follow-up of bladder cancer.

Previously, we showed that murine prothymosin alpha (ProT) enhances the efficacy of a pseudorabies DNA vaccine delivered by bacterial vectors. In this study, we cloned and sequenced the cDNA for porcine ProT. The deduced amino acid sequence of porcine ProT exhibited high homology to ProT from other mammals. Oral Salmonella choleraesuis vaccine carrying the ProT eukaryotic expression plasmid protected mice against virulent S. choleraesuis challenge. The adjuvant effect of ProT on humoral and cellular immune responses enhanced protective efficacy of the vaccine. Furthermore, both humoral and cellular immune responses played roles in the protective immune responses induced by the vaccine. Collectively, our results show that delivery of the ProT gene carried by attenuated S. choleraesuis augmented the immunogenicity of oral S. choleraesuis vaccine.

Background: Polycystic kidney disease (PKD) is a genetic disorder characterized by development of renal cysts and progressive renal dysfunction. Renal tissues from both PKD patients and rodent models of PKD show elevated c-myc expression. Prothymosin alpha (ProT) is positively regulated by c-myc through binding to the E box of its promoter. Through creating transgenic mice and clinical studies, we sought to investigate whether ProT overexpression contributes to PKD development., Methods: ProT heterozygous and homozygous transgenic mice were generated and characterized. Morphologic, histologic, immunohistochemical, and biochemical analyses of the transgenic mice were performed., Results: Two transgenic lines that represented integration at two different loci of the chromosomes were generated. ProT overexpression in the kidneys of homozygous transgenic mice induced a PKD phenotype, which included polycystic kidneys, elevated blood urea nitrogen (BUN), and lethality at about 10 days of age. Similar overexpression pattern of ProT was noted in cystic kidneys of the transgenic mice as well as in human autosomal-recessive PKD (ARPKD) and autosomal-dominant PKD (ADPKD) kidneys. ProT protein levels in the kidneys and urine as well as renal mRNA level of epithelial growth factor receptor (EGFR) of homozygous ProT transgenic mice were significantly higher than heterozygous or nontransgenic littermates. Furthermore, the heterozygous transgenic mice at 17 months of age also developed mild cystic kidneys., Conclusion: Transgenic mice overexpressing ProT represent a novel model for PKD and may provide insights into PKD development. ProT, like c-myc and EGFR, may contribute to the development of renal cysts and may be a potential noninvasive diagnostic molecule of PKD.