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Oxygen is essentially required by most eukaryotic organisms as a scavenger to remove harmful electron and hydrogen ions or as a critical substrate to ensure the proper execution of enzymatic reactions. All nucleated cells can sense oxygen concentration and respond to reduced oxygen availability (hypoxia). When oxygen delivery is disrupted or reduced, the organisms will develop numerous adaptive mechanisms to facilitate cells survived in the hypoxic condition. Normally, such hypoxic response will cease when oxygen level is restored. However, the situation becomes complicated if hypoxic stress persists (chronic hypoxia) or cyclic normoxia-hypoxia phenomenon occurs (intermittent hypoxia). A series of chain reaction-like gene expression cascade, termed hypoxia-mediated gene regulatory network, will be initiated under such prolonged or intermittent hypoxic conditions and subsequently leads to alteration of cellular function and/or behaviors. As a result, irreversible processes occur that may cause physiological disorder or even pathological consequences. A growing body of evidence implicates that hypoxia plays critical roles in the pathogenesis of major causes of mortality including cancer, myocardial ischemia, metabolic diseases, and chronic heart and kidney diseases, and in reproductive diseases such as preeclampsia and endometriosis. This review article will summarize current understandings regarding the molecular mechanism of hypoxia in these common and important diseases.

Fibroblast growth factor 9 (FGF9) promotes cancer progression; however, its role in cell proliferation related to tumorigenesis remains elusive. We investigated how FGF9 affected MA‐10 mouse Leydig tumor cell proliferation and found that FGF9 significantly induced cell proliferation by activating ERK1/2 and retinoblastoma (Rb) phosphorylations within 15 minutes. Subsequently, the expressions of E2F1 and the cell cycle regulators: cyclin D1, cyclin E1 and cyclin‐dependent kinase 4 (CDK4) in G1 phase and cyclin A1, CDK2 and CDK1 in S‐G2/M phases were increased at 12 hours after FGF9 treatment; and cyclin B1 in G2/M phases were induced at 24 hours after FGF9 stimulation, whereas the phosphorylations of p53, p21 and p27 were not affected by FGF9. Moreover, FGF9‐induced effects were inhibited by MEK inhibitor PD98059, indicating FGF9 activated the Rb/E2F pathway to accelerate MA‐10 cell proliferation by activating ERK1/2. Immunoprecipitation assay and ChIP‐quantitative PCR results showed that FGF9‐induced Rb phosphorylation led to the dissociation of Rb‐E2F1 complexes and thereby enhanced the transactivations of E2F1 target genes, Cyclin D1, Cyclin E1 and Cyclin A1. Silencing of FGF receptor 2 (FGFR2) using lentiviral shRNA inhibited FGF9‐induced ERK1/2 phosphorylation and cell proliferation, indicating that FGFR2 is the obligate receptor for FGF9 to bind and activate the signaling pathway in MA‐10 cells. Furthermore, in a severe combined immunodeficiency mouse xenograft model, FGF9 significantly promoted MA‐10 tumor growth, a consequence of increased cell proliferation and decreased apoptosis. Conclusively, FGF9 interacts with FGFR2 to activate ERK1/2, Rb/E2F1 and cell cycle pathways to induce MA‐10 cell proliferation in vitro and tumor growth in vivo. Fibroblast growth factor 9 (FGF9) interacts with FGF receptor 2 (FGFR2) to promote the Rb/E2F1 pathway and cell cycle progression by activating the ERK1/2 pathway. The consequence of FGF9‐induced Rb phosphorylation is the dissociation of the Rb‐E2F1 complexes, the transactivation of E2F1 target genes, such as Cyclin D1, Cyclin E1, and Cyclin A1, cell cycle progression and cell proliferation in MA‐10 cells. In summary, FGF9/FGFR2 activates ERK1/2, Rb/E2F and cell cycle pathways to induce MA‐10 cell proliferation in vitro and MA‐10 tumor growth in vivo.

The DNA-PK maintains cell survival when DNA damage occurs. In addition, aberrant activation of the DNA-PK induces centrosome amplification, suggesting additional roles for this kinase. Here, we showed that the DNA-PK-p53 cascade induced primary cilia formation (ciliogenesis), thus maintaining the DNA damage response under genotoxic stress. Treatment with genotoxic drugs (etoposide, neocarzinostatin, hydroxyurea, or cisplatin) led to ciliogenesis in human retina (RPE1), trophoblast (HTR8), lung (A459), and mouse Leydig progenitor (TM3) cell lines. Upon genotoxic stress, several DNA damage signaling were activated, but only the DNA-PK-p53 cascade contributed to ciliogenesis, as pharmacological inhibition or genetic depletion of this pathway decreased genotoxic stress-induced ciliogenesis. Interestingly, in addition to localizing to the nucleus, activated DNA-PK localized to the base of the primary cilium (mother centriole) and daughter centriole. Genotoxic stress also induced autophagy. Inhibition of autophagy initiation or lysosomal degradation or depletion of ATG7 decreased genotoxic stress-induced ciliogenesis. Besides, inhibition of ciliogenesis by depletion of IFT88 or CEP164 attenuated the genotoxic stress-induced DNA damage response. Thus, our study uncovered the interplay among genotoxic stress, the primary cilium, and the DNA damage response.

Septins are critical for numerous cellular processes through the formation of heteromeric filaments and rings indicating the importance of structural regulators in septin assembly. Several posttranslational modifications (PTMs) mediate the dynamics of septin filaments in yeast. However, little is known about the role of PTMs in regulating mammalian septin assembly, and the in vivo significance of PTMs on mammalian septin assembly and function remains unknown. Here, we showed that SEPT12 was phosphorylated on Ser198 using mass spectrometry, and we generated SEPT12 phosphomimetic knock-in (KI) mice to study its biological significance. The homozygous KI mice displayed poor male fertility due to deformed sperm with defective motility and loss of annulus, a septin-based ring structure. Immunohistochemistry of KI testicular sections suggested that SEPT12 phosphorylation inhibits septin ring assembly during annulus biogenesis. We also observed that SEPT12 was phosphorylated via PKA, and its phosphorylation interfered with SEPT12 polymerization into complexes and filaments. Collectively, our data indicate that SEPT12 phosphorylation inhibits SEPT12 filament formation, leading to loss of the sperm annulus/septin ring and poor male fertility. Thus, we provide the first in vivo genetic evidence characterizing importance of septin phosphorylation in the assembly, cellular function and physiological significance of septins.

Etoposide (ETO) has been used in treating adrenocortical tumor (ACT) cells. Our previous study showed that ETO inhibits ACT cell growth. In the present study, we show that ETO treatment at IC50 (10 μM) inhibited ACT cell growth by inducing cellular senescence rather than apoptosis. Several markers of cellular senescence, including enlarged nuclei, activated senescence-associated β-galactosidase activity, elevated levels of p53 and p21, and down-regulation of Lamin B1, were observed. We further found that ETO induced multiple centrosomes. The inhibition of multiple centrosomes accomplished by treating cells with either roscovitine or centrinone or through the overexpression of NR5A1/SF-1 alleviated ETO-induced senescence, suggesting that ETO triggered senescence via multiple centrosomes. Primary cilia also played a role in ETO-induced senescence. In the mechanism, DNA-PK-Chk2 signaling was activated by ETO treatment; inhibition of this signaling cascade alleviated multiple ETO-induced centrosomes and primary cilia followed by reducing cellular senescence. In addition to DNA damage signaling, autophagy was also triggered by ETO treatment for centrosomal events and senescence. Importantly, the inactivation of DNA-PK-Chk2 signaling reduced ETO-triggered autophagy; however, the inhibition of autophagy did not affect DNA-PK-Chk2 activation. Thus, ETO activated the DNA-PK-Chk2 cascade to facilitate autophagy. The activated autophagy further induced multiple centrosomes and primary cilia followed by triggering senescence.

Acute kidney injury (AKI) is a major and unmet medical need, characterized by a sudden onset of kidney dysfunction that often occurs within 7 days. Adipose-derived stem cells (ADSCs) are known for their regenerative, differentiative, and repair abilities, making them a promising therapeutic option for kidney injury. Platelet-rich fibrin releasate (PRFr), derived from platelet-rich fibrin after static incubation, contains numerous growth factors that may promote the differentiation and proliferation of stem cells. Additionally, acupoints such as Shenshu (BL23) have been used in clinical practice and experimental settings, particularly in renal failure treatments. This study aimed to evaluate the synergistic effects of ADSCs and PRFr, administered separately or in combination, at the Shenshu acupoint (BL23) in New Zealand white rabbits with acute kidney injury. The treatment groups were injected with ADSCs, PRFr, or a combination of both. Serum creatinine (CRE) and blood urea nitrogen (BUN) levels were measured to assess kidney function. Additionally, histological examination of kidney tissue was performed to observe morphological changes and tissue repair. The PRFr + ADSCs treatment group exhibited a significant reduction in CRE and BUN levels during the second week following transplantation. After 7 weeks of treatment, the PRFr + ADSCs group showed the most favorable kidney repair outcomes, with intact glomeruli, no edema or vacuole-like changes in the renal tubular epithelial cells, and no significant infiltration of inflammatory cells in the surrounding tissues. The administration of PRFr, ADSCs, and their combination at the Shenshu acupoint (BL23) demonstrated a potential therapeutic effect in repairing damaged renal cells, improving kidney function, and reducing serum CRE and BUN levels. These findings suggest that injection of PRFr, ADSCs, and their combination at the Shenshu acupoint (BL23) can effectively repair damaged renal cells and improve kidney function in AKI. The observed synergistic effect indicates that this approach holds potential as a novel therapeutic strategy for kidney injury. Further research is needed to optimize treatment protocols and elucidate the underlying mechanisms.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors because of its high metastatic ability. The glutamine (Gln)-deficient microenvironment contributes to PDAC metastasis; however, the underlying molecular mechanisms remain unclear. Here, we demonstrated that melanophilin (MLPH) promotes PDAC metastasis by inducing the regrowth of primary cilia. Using RNA sequencing, we found that MLPH was upregulated in Gln-deficient conditions. MLPH facilitated PDAC metastasis in vitro and in vivo. Clinically, high MLPH expression is positively correlated with metastasis and poor PDAC prognosis. MLPH localized to the centrosome and facilitated the regrowth of primary cilia. The primary ciliogenesis upregulated phospholipase C γ-1 (PLCG1) to promote PDAC metastasis. Interestingly, PLCG1 was localized to the primary cilia, and depletion of PLCG1 alleviated primary ciliogenesis, suggesting a feedforward role for PLCG1 in mediating primary ciliogenesis. Thus, our study revealed a novel function of the MLPH-primary cilia-PLCG1 axis in facilitating PDAC metastasis under Gln deficiency both in vitro and in vivo.

Indium chloride (InCl3), widely used in the electronics and semiconductor industries, has recently been shown to impair testicular development and cause sperm malformations, contributing to male infertility. While testicular Leydig cells are key in testis development and testosterone biosynthesis, the effects of InCl3 on Leydig cell proliferation remain unclear. The present study demonstrated that InCl3 suppresses the proliferation of testicular Leydig cells by disrupting centrosome number regulation. As the centrosome is pivotal in organizing microtubules and forming the mitotic spindle during cell division, InCl3-induced centrosome amplification leads to abnormal spindle formation and genomic instability. Mechanistically, InCl3 increases reactive oxygen species production, resulting in DNA damage and subsequent activation of DNA-dependent protein kinase (DNA-PK), which localizes to the centrosome. Inhibition or knockdown of DNA-PK attenuated InCl3-induced centrosome amplification. Additionally, induction of autophagy was observed upon InCl3 exposure and pharmacological inhibition of autophagy using chloroquine suppressed centrosome amplification. Overall, the present findings reveal that InCl3 inhibits Leydig cell proliferation by promoting centrosome amplification and highlights the involvement of DNA-PK activation and autophagy in this process.

Preeclampsia is a severe gestational hypertensive disorder that may lead to maternal multiple organ dysfunction and adverse fetal outcomes. Aspirin provides a protective effect by reducing the risk of preeclampsia; however, its mechanism of action is unclear. Fibronectin (FN) is a key factor in cell motility and is associated with preeclampsia. Here, we demonstrated that cellular FN expression was elevated in the placenta of preeclamptic patients. The functional roles of plasma and cellular FN in trophoblasts were investigated by treating HTR-8/SVneo cells with exogenous recombinant human FN protein (rhFN) and siRNA, respectively. Trophoblast migration and invasion were inhibited by rhFN and facilitated by FN knockdown. Moreover, rhFN activated ERK and Akt signaling in trophoblasts, and FN-suppressed cell motility was rescued by ERK and/or Akt inhibitors. In this study, aspirin suppressed trophoblast cellular FN expression and reversed FN-mediated cell functions, including cell migration, invasion, and ERK/Akt signal changes. Taken together, the results of this study revealed the effects of FN on trophoblast motility and signaling; aspirin inhibits FN expression and reverses FN-mediated trophoblast biology. These results provide a drug mechanism for disease prevention and a target for preeclampsia intervention.

Background Infertility affects one in six individuals worldwide despite the advancement of assisted reproductive technologies. Successful embryo implantation is the first step of pregnancy, which relies on the establishment of a receptive uterine microenvironment. However, the mechanisms governing uterine receptivity and implantation failure remain incompletely characterized. Primary cilia serve as key cellular signaling hubs, yet their contribution to human decidualization and uterine receptivity remains largely unexplored. Methods Primary cultured human endometrial stromal cells (ESCs) were used to investigate the mechanisms of decidualization, functions of primary cilia, and effects of transforming growth factor-β (TGF-β) in inhibiting prostaglandin E 2 (PGE 2 )-induced decidualization. Human endometrial tissues (n = 108) were used to evaluate the clinicopathological parameters. The percentage of ciliated cells and cilia length were determined by immunofluorescent staining and AI-assisted quantification. Pseudopregnancy and pregnancy mouse models were employed to assess the effects of TGF-β1 on uterine receptivity and implantation outcomes. Results Prostaglandin E 2 , through binding to the EP4 receptor located at the primary cilium, stimulates ESC decidualization, which is augmented by 17β-estradiol and progesterone. Loss of ciliogenesis by genetic or pharmacological inhibition impairs decidualization. Proinflammatory cytokines such as TGF-β inhibit ciliogenesis and thus markedly attenuate PGE 2 -mediated decidualization. Mechanistically, TGF-β1 suppressed chicken ovalbumin upstream promoter transcription factor II and its downstream effector kinesin family member 3B, thereby inhibiting ciliogenesis and PGE₂-EP4 signaling. In mice, intrauterine administration of TGF-β1 impaired implantation, while TGF-β receptor blockade restored ciliogenesis, decidualization, and fertility. In women with endometriosis, ESCs displayed shortened cilia and reduced decidual response, which are due to elevated uterine and peritoneal TGF-β1-mediated suppression of ciliogenesis. Finally, women who failed to conceive after in vitro fertilization-embryo transfer (IVF-ET) have shorter and fewer primary cilia in ESCs. Receiver operating characteristic curve analysis demonstrated that both cilia length (AUC = 0.86) and ciliation frequency (AUC = 0.79) can serve as biomarkers for endometrial receptivity, providing predictive value for reproductive outcomes independent of ovarian reserve. Conclusions Endometrial primary cilia are indispensable for decidualization and are potential biomarkers for predicting endometrial receptivity. Targeting TGF-β signaling to restore ciliated cell number and ciliary length may serve as a potential therapeutic strategy to improve fertility outcomes.