American journal of physiology. Cell physiology最新文献

The disease burden of renal cell carcinoma (RCC) has decreased in recent years with advances in treatment, but its pathogeny still remains elusive. We aim to study the role of homeobox A3 (HOXA3)/ubiquitin-specific peptidase 15 (USP15)/SQSTM1 axis on autophagy and M2-type macrophage polarization in RCC. In this study, cell apoptosis and proliferation were assessed by flow cytometry and CCK-8. Autolysosome fusion was observed by immunofluorescence detection of LC3 and LAMP2. The binding between HOXA3 and USP15 promoter was tested by chromatin immunoprecipitation (ChIP), EMSA, and dual-luciferase reporter assays. Also, the interaction between deubiquitinated enzyme (DUB) USP15 and SQSTM1, and ubiquitinated level of SQSTM1 were determined by co-immunoprecipitation (Co-IP) assay. Expression levels of HOXA3, USP15, C-C motif chemokine 2 (CCL2), CCL2 receptor (CCR2), M2-type macrophages, and autophagy-related markers were measured by Western blot, quantitative reverse transcription PCR (RT-qPCR), ELISA, and immunohistochemistry. Role of HOXA3/USP15 axis was verified by xenograft tumor experiment in vivo. We showed upregulated HOXA3 in RCC tissues and cells, and RCC tissues with metastasis showed higher HOXA3 level. The higher HOXA3 expression was relevant to worse overall survival in patients with RCC. HOXA3 induced RCC cell proliferation, and suppressed autophagy and apoptosis via transcriptionally activating USP15 expression. USP15 then induced deubiquitination modification of SQSTM1 in RCC cells. SQSTM1 supported M2-type macrophage polarization by inducing CCL2 secretion. HOXA3 or USP15 knockdown suppressed tumor growth and M2-type macrophage infiltration in vivo. In conclusion, HOXA3 transcriptionally activates USP15 expression, and upregulated USP15 facilitates the deubiquitination of SQSTM1 in RCC. This process on the one hand suppresses autophagy, on the other hand increases M2-type macrophage polarization through stimulating the secretion of CCL2.NEW & NOTEWORTHY We report a novel finding that highly expressed homeobox A3 (HOXA3) transcriptionally activates the expression of ubiquitin-specific peptidase 15 (USP15), resulting in the promotion of deubiquitination of SQSTM1. This process on the one hand suppresses autophagy in renal cell carcinoma (RCC), on the other hand increases M2-type macrophage polarization in the tumor microenvironment through stimulating the secretion of C-C motif chemokine 2 (CCL2).

Transfer RNA-derived small RNAs (tsRNAs), a recently identified noncoding RNA subset, are mainly classified into transfer RNA (tRNA)-derived small RNA fragments (tRFs) and tRNA-derived stress-induced RNAs (tiRNAs). tsRNAs dysregulation is frequently observed in numerous cancer types, suggesting involvement in tumorigenesis. However, their functions in breast cancer (BC) remain to be fully understood. Here, it was discovered that tRF-33-MEF91SS2PMFI0Q (tRF-33), derived from mature tRNA-Lys^TTT, was markedly upregulated in human epidermal receptor 2 (HER2)-negative BC cells and tissue samples. tRF-33 stimulated the proliferation, migration, and invasiveness of BC cells in vitro and facilitated tumor progression in vivo. Mechanistically, tRF-33 was found for the first time to bind directly to the 3'-UTR of IGF1, resulting in downregulation of both its mRNA and protein and thus affecting mitochondrial homeostasis and progression of BC. These results demonstrate a novel tsRNA modulatory mechanism and a potential direction for treating HER2-negative BC.NEW & NOTEWORTHY In this study, we identified differential expression of tRNA fragments in HER2-negative BC tissues compared with adjacent normal tissues, observing significant upregulation of an i-tRF type tRF-33-MEF91SS2PMFI0Q (tRF-33) in the tumor tissue. We also found that tRF-33 promoted tumorigenesis in BC cells. We demonstrated for the first time that IGF1 was a target gene of tRF-33 and also showed that the tRF-33/IGF1 axis impaired mitochondrial dynamics, thus affecting mitochondrial homeostasis and promoting HER2-negative BC progression.

Many pathogens including viruses enter cells by endocytosis. We identified and evaluated novel endocytosis inhibitors capable of blocking the entry of the HIV-1 Transactivation of Transcription protein (Tat) protein into neuronal cells and investigated their potential protective properties against Tat-induced neurotoxicity. In this study, the compounds Les-6631 and Les-6633 were synthesized and assessed. The effects of these compounds on the internalization of dextran and the cell-penetrating peptide (CPP) Tat-Cy5 complex in nerve cells were examined. In addition, the ability of these compounds to protect against oxidative stress and DNA damage induced by the full-length Tat protein was investigated. Les-6631 and Les-6633 were found to inhibit endocytosis better than the classical endocytosis inhibitor chlorpromazine, thereby effectively preventing the entry of the Tat protein into nerve cells. Moreover, compounds demonstrated the capacity to reduce oxidative stress and protect DNA from Tat-induced damage. In a neuro-AIDS model, both compounds proved effective in preventing neurotoxicity associated with HIV-1 infection, indicating its potential for therapeutic applications. Les-6631 and Les-6633 thus can protect cells from the harmful effects of pathogens. Their use in a neuro-AIDS model suggests a potential application in protective therapies for the nervous system in patients with HIV.NEW & NOTEWORTHY This study identifies novel rhodadyn-based inhibitors, Les-6631 and Les-6633, which selectively block dynamin's GTPase activity while sparing clathrin-mediated pathways. They effectively inhibit cellular uptake, protect neural cells from HIV-1 Tat-induced oxidative stress, and reduce mitochondrial and DNA damage. Their selective dynamin inhibition and antioxidant properties highlight their therapeutic potential for neurodegeneration and viral infections, offering cell protection without disrupting essential endocytic functions.

Filamin A (FLNA) is an actin-binding protein that has been reported to interact with STIM1 modulating the activation of Orai1 channels. Cleaving of FLNA by calpain leads to a C-terminal fragment that is involved in a variety of functional and pathological events, including pro-oncogenic activity in different types of cancer. Here, we show that full-length FLNA is downregulated in samples from patients with colon cancer as well as in the adenocarcinoma cell line HT-29. This is consistent with an increased calpain-dependent FLNA cleaving with enhanced expression of the C-terminal FLNA fragment accompanied by enhanced expression of Orai1 and STIM1, as well as store-operated Ca²⁺ entry (SOCE). To further explore the mechanism underlying the enhancement of SOCE by the C-terminal FLNA fragment, we expressed in HEK-293 cells the C-terminal FLNA region encompassing repeats 16-24 (FLNA^16-24 fragment), which enhanced both Orai1 and STIM1 as well as SOCE. Transfection of the FLNA^16-24 fragment attenuates Orai1 and STIM1 protein degradation, and, specifically, abrogates Orai1α lysosomal degradation and retains this channel in the plasma membrane. However, the C-terminal FLNA fragment did not induce a detectable modification in Orai1β degradation. Due to the relevance of SOCE in cell physiology, our results provide evidence of a novel mechanism for the regulation of Ca²⁺ influx with relevant pathophysiological implications.NOTE & NOTEWORTHY FLNA cleaving by calpain has been observed in a variety of tumoral, including prostate and colorectal cancer cells, as well as in nontumoral cells, leading to a C-terminal fragment encompassing repeats 16-24. Expression of the FLNA^16-24 fragment in HEK-293 cells enhances Orai1 and STIM1 expression, as well as SOCE, a mechanism mediated by attenuation of Orai1α and STIM1 degradation, providing evidence for a novel mechanism for the regulation of SOCE in normal and malignant cells.

Pulmonary ionocytes express high levels of cystic fibrosis transmembrane conductance regulator (CFTR) channels. When studied using the short-circuit current technique, ionocytes produce CFTR-dependent short-circuit currents consistent with Cl^- secretion. However, when studied without a voltage clamp, data indicate that ionocytes absorb Cl^-. In this review, we resolve these seemingly conflicting findings by considering the different transepithelial voltages and the resultant movement of Cl^- during short circuit and physiological open-circuit conditions. This analysis indicates that behavior under short-circuit conditions cannot be directly extrapolated to infer behavior under physiologic conditions. Finally, we discuss the potential role of basolateral Cl^- channels in controlling absorption and secretion in ionocytes.

In this study, we used an ex vivo-in vitro model to assess the effect of feeding older (50-70 yr) adults a casein protein hydrolysate (CPH) compared with nonbioactive nonessential amino acid (NEAA) supplement on muscle protein synthesis (MPS) and markers of muscle protein breakdown (MPB). As a secondary objective, to assess any attenuation with aging, we compared the anabolic response to CPH-fed serum from older and young adults. Serum from seven healthy older and seven young men following overnight fast and 60-min postprandial ingestion of CPH or NEAA (0.33 g·kg^-1 body mass) was used to condition C2C12 myotube media. Analysis by two-way ANOVA of the fed relative to fasted MPS response revealed a main effect for protein type in pmTOR (P = 0.009), p70S6K (P = 0.031), p4E-BP1 (P = 0.047), and MPS (P = 0.041) with a greater response to CPH-fed serum, and interaction effects (age × protein) between young and old serum for pmTOR (P = 0.009) and p70S6K (P = 0.016). In addition, significant changes in myotube diameter (P = 0.049), atrogin-1 (P = 0.004), and MuRF-1 (P = 0.012) in response to CPH-fed compared with fasted serum were observed with no differences between young and old serum. In conclusion, this study demonstrated that CPH-fed serum from both young and older (50-70 yr) adults can stimulate MPS and muscle growth and can suppress biomarkers of muscle protein breakdown processes.NEW & NOTEWORTHY This study extended previously developed coculture models and found that treating skeletal muscle cells with ex vivo human serum following feeding with a casein protein hydrolysate resulted in greater protein signaling, muscle protein synthesis, muscle growth, and lower expression of genes related to muscle protein breakdown compared with feeding with a nonessential amino acid control. These findings were similar using serum from young and older adults.

Hyperglycemia and hyperglycosuria, two primary characteristics of diabetes mellitus, may increase the risk of cancer initiation, particularly for bladder cancer. The effectiveness of metformin, a common antidiabetic agent, is determined by its ability to induce growth differentiation factor 15 (GDF15). However, the mechanism of the GDF15 in relation to glucose, which influences the tumor microenvironment in the human bladder, is not fully understood. This study explores the potential roles of GDF15 in response to glucose in the human bladder. High glucose treatment (30 mM) enhanced phosphorylation of AKT at S473 and AMP-activated protein kinase α1/2 (AMPKα1/2) at S485 to block the counteracting effect of metformin on the AMPK activity in bladder cancer and stroma [human bladder stromal fibroblast (HBdSF) and human bladder smooth muscle cell (HBdSMC)] cells compared with normal glucose treatment (5 mM). Metformin modulated the expressions of GDF15, NDRG1, Maspin, and epithelial-to-mesenchymal transition (EMT) markers to attenuate cell proliferation and invasion of bladder cancer cells. Caffeic acid phenethyl ester (CAPE), like metformin, behaves as an inducer of AMPK activity to stimulate GDF15 expression. Knockdown of GDF15 blocked the downregulation of CAPE on the contraction of HBdSMCs. Both CAPE-induced GDF15 expression and the supernatant from bladder cancer cells with overexpressing GDF15 impeded the HBdSF and HBdSMC migration, suggesting that CAPE-upregulated GDF15 blocked the cell migration. These findings reveal that high glucose treatment inhibits the counteracting effects of either metformin or CAPE on the AMPK activity and GDF15 is downregulated by glucose and induced by metformin and CAPE in both stroma and cancer cells. Furthermore, GDF15 is an antitumor gene facilitating communication between stroma and cancer cells in the human bladder.NEW & NOTEWORTHY This study investigates the counteraction of either CAPE or metformin with the AMPK activity increasing GDF15 expression in human bladder cells. The findings are the first study to indicate the secretion of GDF15 from cancer and stroma cells via autocrine or paracrine mechanisms. Our study suggests that GDF15, an antitumor gene in the human bladder induced by AMPK inducers, acts as a communication link between stroma and cancer cells in the human bladder.

Over the past few decades, the primary cilium, an inconspicuous cell organelle, has increasingly become the focus of current research. The primary cilium is a microtubule-based, nonmotile, antenna-like structure that is present in almost all mammalian cells. The ciliary membrane incorporates a large number of receptor molecules, which further characterize this cellular organelle. These include receptors of the Sonic hedgehog (Shh)-, Wnt-, or platelet-derived growth factor (PDGF) signaling pathways. For this reason, as well as due to the fact that extracellular signaling molecules can bind to the ciliary membrane, primary cilia have been named "the antenna of the cell." In addition to their signaling function, the association of ciliary dysfunctions with a variety of diseases, so-called ciliopathies, underscores the importance of this functional cellular structure. Recent studies have also implicated primary cilia in the adaptation to low-oxygen conditions, which are characteristic of ischemia, such as in stroke or myocardial infarction, or tumor entities. The aim of this review is to provide an overview of these multiple facets and to take a closer look at the evolution of an inconspicuous cell organelle to a major player in hypoxia.

Long noncoding RNA (lncRNA) and N6-methyladenosine (m6A) methylation modification have recently been suggested as potential functional modulators in ovarian endometriosis, however, the function and mechanism of m6A-modified lncRNA in ovarian endometriosis remain poorly understood. In this study, we demonstrated that lncRNA UBOX5-AS1 expression was significantly elevated in ovarian endometriosis tissue and primary ectopic endometrial stromal cells. The expression of lncRNA UBOX5-AS1, which has m6A modifications, was highly positively correlated with demethylase Alk B homologous protein 5 (ALKBH5) expression and autophagy. Functional studies revealed that increased ALKBH5 and lncRNA UBOX5-AS1 expression promoted cell autophagy, proliferation, and invasion in endometriosis in vitro. LncRNA UBOX5-AS1 mediates ALKBH5-regulated autophagy, proliferation, and invasion. ALKBH5-mediated autophagy facilitates cell proliferation, migration, and invasion. In vivo, the knockdown of ALKBH5 inhibited endometriotic lesion growth. Mechanistically, we observed that ALKBH5 mediated the m6A demethylation of lncRNA UBOX5-AS1 and promoted its expression. Thus, our findings highlight that ALKBH5/lncRNA UBOX5-AS1 might serve as potential targets for ovarian endometriosis therapy in the future.NEW & NOTEWORTHY In the present study, we investigated the role and potential molecular mechanism of long noncoding RNA (lncRNA) UBOX5-AS1 in ovarian endometriosis progression. Combined with the aforementioned, we proposed the hypothesis that lncRNA UBOX5-AS1 regulated by Alk B homologous protein 5 (ALKBH5)-mediated N6-methyladenosine (m6A) modification contributes to the progression of ovarian endometriosis progression.

The optimum length for force generation (L₀) increases as activation is reduced, challenging classic theories of muscle contraction. Although the activation dependence of L₀ is seemingly consistent with length-dependent Ca²⁺ sensitivity, this mechanism cannot explain the apparent force dependence of L₀ or the effect of series compliance on activation-related shifts in L₀. We have tested a theory proposing that the activation dependence of L₀ relates to force depression resulting from shortening against series elasticity. This theory predicts that significant series compliance would cause tetanic L₀ to be shorter than the length corresponding to optimal filament overlap, thereby increasing the activation dependence of L₀. We tested this prediction by determining L₀ and maximum tetanic force (P₀) with (L_{0_spring}, P_{0_spring}) and without added compliance in bullfrog semitendinosus muscles. The activation dependence of L₀ was characterized with the addition of twitch and doublet contractions. Springs attached to muscles gave added fixed-end compliances of 11%-39% and induced force depression for tetanic fixed-end contractions (P_{0_spring} < P₀). We found strong, negative correlations between spring compliance and both P_{0_spring} (r² = 0.89-0.91) and L_{0_spring} (r² = 0.60-0.63; P < 0.001), whereas the activation dependence of L₀ was positively correlated to added compliance (r² = 0.45, P = 0.011). However, since the compliance-mediated reduction in L₀ was modest relative to the activation-related shift reported for the bullfrog plantaris muscle, additional factors must be considered. Our demonstration of force depression under novel conditions adds support to the involvement of a stress-induced inhibition of cross-bridge binding.NEW & NOTEWORTHY Length-dependent Ca²⁺ sensitivity does not fully explain the activation dependence of optimum length (L₀). We demonstrate using an isolated muscle preparation and added series compliance that substantial force depression can arise during an isometric contraction, causing tetanic L₀ to shift to a shorter length. Our findings illustrate that series compliance, via the work and length dependencies of force depression, partially uncouples force generation from myofilament overlap, which ultimately increases the activation (or force) dependence of L₀.