Insulin-like growth factor binding protein 5 accelerate the senescence of periodontal ligament stem cells.

Evidences have showed stem cell mediated tissue regeneration is a promising method for the treatment of periodontitis. Insulin-like growth factor binding proteins-5 (IGFBP5) is a member of the insulin growth factor (IGFs) family and plays a regulatory role in cell proliferation and differentiation. Our previous study showed that IGFBP5 can promote osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and enhance periodontal tissue regeneration mediated by PDLSCs. However, the function of IGFBP5 in the process of PDLSCs senescence remains unclear. The present study showed IGFBP5 mRNA level was highly expressed in passage-induced aged PDLSCs cells. IGFBP5 knockdown decreased the ratio of senescence associated ß-galactosidase (SA-ß-Gal) positive cells, enhanced the activity of TERT, and down-regulated the expression levels of P16, P21, P53 mRNA and protein. Overexpression of IGFBP5 increased the ratio of SA-ß-Gal positive staining PDLSCs, decreased the activity of telomerase TERT, and up-regulated the expression levels of P16, P21, P53 mRNA and protein related to PDLSCs senescence. In conclusion, IGFBP5 can accelerate the senescence of PDLSCs, indicating the potential target for maintaining the "young state" of stem cells.

Exosome-Encapsulated microRNA-140-5p Alleviates Neuronal Injury Following Subarachnoid Hemorrhage by Regulating IGFBP5-Mediated PI3K/AKT Signaling Pathway.

Recent literature has highlighted the therapeutic implication of exosomes (Exos) released by adipose tissue-originated stromal cells (ADSCs) in regenerative medicine. Herein, the current study sought to examine the potential protective effects of ADSC-Exos on neuronal injury following subarachnoid hemorrhage (SAH) by delivering miR-140-5p. Firstly, isolated primary neurons were co-cultured together with well-identified ADSC-Exos. TDP-43-treated neurons were subsequently treated with PKH67-ADSC-Exos and Cy3-miR-140-5p to assess whether ADSC-Exos could transmit miR-140-5p to the recipient neurons to affect their behaviors. Moreover, a luciferase assay was carried out to identify the presumable binding of miR-140-5p to IGFBP5. IGFBP5 rescue experimentation was also performed to testify whether IGFBP5 conferred the impact of miR-140-5p on neuronal damage. The role of PI3K/AKT signaling pathway was further analyzed with the application of its inhibitor miltefosine. Lastly, SAH rat models were developed for in vivo validation. It was found that ADSC-Exos conferred protection against TDP-43-caused neuronal injury by augmenting viability and suppressing cell apoptosis. In addition, miR-140-5p was transmitted from ADSC-Exos to neurons and post-transcriptionally downregulated the expression of IGFBP5. As a result, by means of suppressing IGFBP5 and activating the PI3K/AKT signaling pathway, miR-140-5p from ADSC-Exos induced a neuroprotective effect. Furthermore, in vivo findings substantiated the aforementioned protective role of ADSC-Exos-miR-140-5p, contributing to protection against SAH-caused neurological dysfunction. Collectively, our findings indicated that ADSC-Exos-miR-140-5p could inhibit TDP-43-induced neuronal injury and attenuate neurological dysfunction of SAH rats by inhibiting IGFBP5 and activating the PI3K/Akt signaling pathway.

Biological effects and regulation of IGFBP5 in breast cancer.

The insulin-like growth factor receptor (IGF1R) pathway plays an important role in cancer progression. In breast cancer, the IGF1R pathway is linked to estrogen-dependent signaling. Regulation of IGF1R activity is complex and involves the actions of its ligands IGF1 and IGF2 and those of IGF-binding proteins (IGFBPs). Six IGFBPs are known that share the ability to form complexes with the IGFs, by which they control the bioavailability of these ligands. Besides, each of the IGFBPs have specific features. In this review, the focus lies on the biological effects and regulation of IGFBP5 in breast cancer. In breast cancer, estrogen is a critical regulator of IGFBP5 transcription. It exerts its effect through an intergenic enhancer loop that is part of the chromosomal breast cancer susceptibility region 2q35. The biological effects of IGFBP5 depend upon the cellular context. By inhibiting or promoting IGF1R signaling, IGFBP5 can either act as a tumor suppressor or promoter. Additionally, IGFBP5 possesses IGF-independent activities, which contribute to the complexity by which IGFBP5 interferes with cancer cell behavior.

microRNA-181a Promotes Mitochondrial Dysfunction and Inflammatory Reaction in a Rat Model of Intensive Care Unit-Acquired Weakness by Inhibiting IGFBP5 Expression.

This study investigated mechanisms by which microRNA (miR)-181a orchestrates mitochondrial dysfunction and inflammation in a rat model of intensive care unit-acquired weakness (ICU-AW). Expression of miR-181a and insulin-like growth factor binding protein 5 (IGFBP5) was detected and then miR-181a was overexpressed or inhibited and IGFBP5 was overexpressed in the ICU-AW rats. The expression of UCP-3, metaphase chromosome protein 1 (MCP1), mitochondrial DNA (mtDNA), inflammatory factors, phosphorylation (p)-JAK1, p-STAT1, and p-STAT2 were measured in skeletal muscle tissues; binding of miR-181a to IGFBP5 was evaluated by a dual-luciferase reporter assay. The results demonstrated high expression of miR-181a and low expression of IGFBP5 in ICU-AW versus control rats; IGFBP5 was identified as a target gene of miR-181a. Further experiments demonstrated that ICU-AW rats suffered from marked loss of grip strength and decreased adenosine triphosphate production, mtDNA content, and UCP-3 mRNA expression in skeletal muscles; this was accompanied by elevated TNF-α, IL-6, IL-1ß, MCP1, p-JAK1, p-STAT1, and p-STAT2 levels. Importantly, miR-181a suppression alleviated strength loss, inflammatory reaction, and mitochondrial dysfunction and diminished the phosphorylation levels of JAK1, STAT1, and STAT2 whereas IGFBP5 upregulation rescued the effect of miR-181a overexpression in ICU-AW rats. These results indicate that miR-181a promotes mitochondrial dysfunction and inflammation by activating the JAK/STAT pathway via IGFBP5 in ICU-AW model rats.

hsa_circ_0058122 knockdown prevents steroid-induced osteonecrosis of the femoral head by inhibiting human umbilical vein endothelial cells apoptosis via the miR-7974/IGFBP5 axis.

BACKGROUND: Steroid-induced osteonecrosis of femoral head (SONFH) is a serious complication of glucocorticoid overused. Recent evidence has demonstrated that circRNAs exert key pathophysiological roles in a variety of disease processes. However, the role of circRNA in SONFH remains largely unknown. The current study sought to evaluate how hsa_circ_0058122 affects SONFH in dexamethasone (DEX) treated human umbilical vein endothelial cells (HUVECs) model. METHODS: RT-PCR was used to demonstrate the hsa_circ_0058122 expression level in Dex-treated HUVECs cells. The effects of hsa_circ_0058122 on HUVECs apoptosis were evaluated via overexpression plasmid and siRNA. Using dual-luciferase and fluorescence in situ hybridization assays, we demonstrated that hsa_circ_0058122 binds to miR-7974 thereby facilitating HUVECs apoptosis. Bioinformatics analysis and western blot were performed to confirm target genes of hsa-miR-7974. RESULTS: In our previous work, we revealed the top 20 elevated circRNAs in SONFH patients were hsa_circ_0010027, hsa_circ_0058115, hsa_circ_0010026, hsa_circ_0058839, hsa_circ_0056886, hsa_circ_0056885, hsa_circ_0058146, hsa_circ_0058105, hsa_circ_0058112, hsa_circ_0058143, hsa_circ_0058102, hsa_circ_0058090, hsa_circ_0075353, hsa_circ_0058126, hsa_circ_0058130, hsa_circ_0058140, hsa_circ_0058122, hsa_circ_0058123, hsa_circ_0058103, and hsa_circ_0058121. Among these, hsa_circ_0058122 was finally selected for further investigation. We found hsa_circ_0058122 expression was markedly elevated in Dex-treated HUVECs cells, and the Dex-mediated HUVEC apoptosis was impaired in hsa_circ_0058122-silenced cells and increased in hsa_circ_0058122-overexpressing cells. hsa_circ_0058122 competitively binds to hsa-miR-7974, which in turn interacts with insulin-like growth factor binding protein 5 (IGFBP5). CONCLUSIONS: hsa_circ_0058122/miR-7974/IGFBP5 was proposed to be a key regulatory pathway for SONFH. DEX treatment upregulated hsa_circ_0058122 expression in HUVECs, which sponged miR-7974, thereby increasing IGFBP5 expression, the hsa_circ_0058122/miR-7974/IGFBP5 axis contributed to the Dex-mediated apoptosis. These findings may identify novel targets for SONFH molecular therapy.

Overexpression of long noncoding RNA MCM3AP-AS1 promotes osteogenic differentiation of dental pulp stem cells via miR-143-3p/IGFBP5 axis.

MCM3AP-AS1 regulates the cartilage repair in osteoarthritis, but how it regulates osteogenic differentiation of dental pulp stem cells (DPSCs) remains to be determined. DPSCs were isolated and induced for osteogenic differentiation. MCM3AP-AS1 expression was increased along with the osteogenic differentiation of DPSCs, whose expression was positive correlated with those of OCN, alkaline phosphatase (ALP) and RUNX2. On contrary, miR-143-3p expression was decreased along with the osteogenic differentiation and was negatively correlated with those of OCN, ALP and RUNX2. Dual-luciferase reporter gene assay showed that miR-143-3p can be negatively regulated by MCM3AP-AS1 and can regulate IGFBP5. MCM3AP-AS1 overexpression increased the expression levels of osteogenesis-specific genes, ALP activity and mineralized nodules during DPSC osteogenic differentiation, while IGFBP5 knockdown or miR-143-3p overexpression counteracted the effect of MCM3AP-AS1 overexpression in DPSCs. Therefore, this study demonstrated the role of MCM3AP-AS1/miR-143-3p/IGFBP5 axis in regulating DPSC osteogenic differentiation.

LncRNA HOXA11-AS aggravates the keloid formation by targeting miR-148b-3p/IGFBP5 axis.

Long non-coding RNA (lncRNA) homeobox (HOX) A11 antisense (HOXA11-AS) mediates cell-biological phenotypes of keloid fibroblasts and influence the keloid progression, yet the underlying mechanism need to be further understood. HOXA11-AS, microRNA miR-148b-3p and Insulin like growth factor binding protein 5 (IGFBP5) expression were detected by RT-qPCR or western blot. CCK-8 and colony formation assays were applied to examine the cell proliferation. The cell migration was determined via Transwell migration assays. The cell apoptosis was determined by western blots with anti-Bax antibodies and anti-Cleaved Caspase-3 antibodies. The interplay between miR-148b-3p, HOXA11-AS and IGFBP5 was confirmed by luciferase reporter or RNA immunoprecipitation assay. The amplification of HOXA11-AS and IGFBP5 was detected in keloid and keloid fibroblasts, while miR-148b-3p expression was reduced. Moreover, downregulation of HOXA11-AS in keloid fibroblasts inhibited cell proliferation, migration and triggered apoptosis. Mechanically, HOXA11-AS was proved to sponge miR-148b-3p and abrogate the inhibition on miR-148b-3p target, IGFBP5 mRNA, thus promoting keloid fibroblasts proliferation, migration and inhibiting apoptosis. These results find that HOXA11-AS promotes keloid progression by miR-148b-3p/IGFBP5 axis, suggesting the potential of targeting HOXA11-AS/miR-148b-3p/IGFBP5 axis to combat keloid.

Insulin-Like Growth Factor Binding Protein 5: an Important Regulator of Early Osteogenic Differentiation of hMSCs.

Insulin-like growth factor binding protein 5 (IGFBP5) is broadly bioactive, but its role in osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) remains to be clarified. Here, we demonstrated that IGFBP5 expression was markedly increased during the early osteogenic differentiation of hMSCs. We then over-expressed and knocked down this gene in hMSCs and evaluated the impact of manipulation of IGFBP5 expression on osteogenic differentiation based upon functional assays, ALP staining, and expression of osteogenic markers. Together, these analyses revealed that IGFBP5 over-expression enhanced early osteogenic differentiation, as evidenced by increased ALP staining and osteogenic marker induction, whereas knocking down this gene impaired the osteogenic process. Over-expression of IGFBP5 also markedly bolstered the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation level, while IGFBP5 knockdown suppressed this signalling activity. We additionally compared the impact of simultaneous IGFBP5 overexpression and ERK1/2 inhibitor treatment to the effect of IGFBP5 over-expression alone in these hMSCs, revealing that small molecule-mediated EKR1/2 inhibition was sufficient to impair osteogenic differentiation in the context of elevated IGFBP5 levels. These findings indicated that IGFBP5 drives the early osteogenic differentiation of hMSCs via the ERK1/2 signalling pathway. Our results offer value as a foundation for future efforts to study and treat serious bone-related diseases including osteoporosis.

Human-derived NLS enhance the gene transfer efficiency of chitosan.

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA. In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5. These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.

Significant association of serum autoantibodies to TYMS, HAPLN1 and IGFBP5 with early stage canine malignant mammary tumours.

Canine mammary tumours (CMTs) are the most prevalent neoplasms in female dogs. Despite the high incidence of such tumours, a lack of easily accessible biomarkers still impedes early diagnosis of malignant CMTs. Herein we identify thymidylate synthetase (TYMS), hyaluronan and proteoglycan link protein 1 (HAPLN1) and insulin-like growth factor-binding protein 5 (IGFBP5) as CMT antigens eliciting corresponding autoantibodies in CMT cases. We establish enzyme-linked immunosorbent assays (ELISAs) to detect autoantibodies to TYMS (TYMS-AAb), HAPLN1 (HAPLN1-AAb) and IGFBP5 (IGFBP5-AAb) in sera from 81 dogs with malignant CMTs (41 in Stage I), 24 with benign CMTs and 35 healthy controls. Levels of all the three autoantibodies are elevated in the malignant group compared with the healthy or the benign group; notably, the elevated autoantibody levels significantly correlate with the stage-I CMTs. For discriminating malignant CMTs from healthy control, the area under curve (AUC) of TYMS-AAb is 0.694 with specificity of 82.9% and sensitivity of 50.6%. The AUC of utilising HAPLN1-AAb for distinguishing the stage-I CMTs from healthy controls is 0.711 with specificity of 77.1% and sensitivity of 58.5%. In differentiating malignant CMTs from the benign, the AUC of IGFBP5-AAb reaches 0.696 with specificity of 70.8% and sensitivity of 67.9%, and a combination of IGFBP5-AAb and TYMS-AAb increases the AUC to 0.72. Finally, the AUC of combined HAPLN1-AAb and IGFBP5-AAb in discriminating the stage-I CMTs from the benign achieves 0.731. Collectively, this study highlights a significant association of the three serum autoantibodies with early stage malignant CMTs.

Homocysteine downregulates cardiac homeobox transcription factor NKX2.5 via IGFBP5.

Homocysteine (Hcy) is an independent risk factor of congenital heart disease (CHD), but its exact underlying mechanism is unclear. In this study, we collected amniotic fluid (AF) supernatant samples from pregnant women carrying CHD-affected (n = 16) or normal (n = 16) fetuses. We found that Hcy concentrations were higher in the AF of the CHD group when compared with normal pregnancies. Also, Western blot showed that NK2 homeobox 5 (NKX2.5) was decreased and insulin-like growth factor binding protein 5 (IGFBP5) was increased in the AF of the CHD group. In the H9C2 cell culture experiment, 500 µmol/L Hcy downregulated NKX2.5 and upregulated IGFBP5. Real-time PCR and Western blot showed that NKX2.5 expression was reduced in H9C2 cells treated with IGFBP5. Luciferase reporter gene demonstrated that IGFBP5 decreased the transcription of the NKX2.5 promoter. Chromatin immunoprecipitation and electrophoretic mobility shift assay suggested that IGFBP5 binds to the NKX2.5 promoter region. Thus, the data indicated that one of the possible mechanisms by which Hcy is involved in CHD may be that Hcy inhibits NKX2.5 expression partly through IGFBP5.NEW & NOTEWORTHY We found that Hcy and IGFBP5 were increased, whereas NKX2.5 was decreased, in AF of CHD. Meanwhile, Hcy could upregulate IGFBP5 but downregulate NKX2.5, and IGFBP5 inhibited NKX2.5 expression in vitro. Moreover, IGFBP5 can bind to the NKX2.5 promoter region and reduce NKX2.5 transcriptional activity.

MiR-193b inhibits autophagy and apoptosis by targeting IGFBP5 in high glucose-induced trophoblasts.

INTRODUCTION: Inhibiting apoptosis of trophoblasts in women with gestational diabetes mellitus (GDM) is expected to guarantee adequate nutrition for the fetus and avoid abortion. MiR-193b is one of the most downregulated miRNAs in GDM patients. However, less is known about the role of miR-193b in autophagy and apoptosis in GDM patients. METHODS: We detected the expression of miR-193b in GDM patients. Then, we cultured human trophoblasts (HTR8 cells) with high glucose (HG) to simulate a diabetic environment in vitro, and further explored the effects of miR-193b on apoptosis and autophagy of HG-treated HTR8 cells. RESULTS: The expression of miR-193b was significantly downregulated in the peripheral blood of GDM patients compared with healthy controls, and decreased miR-193b caused apparent autophagy and a substantially high apoptosis rate in HG-treated HTR8 cells. These effects were reversed by enhancing miR-193b expression or using the autophagy inhibitor 3-MA. Inhibiting miR-193b induced the pro-autophagic, cytostatic, and pro-apoptotic effects reduced by 3-MA in HTR8 cells upon HG treatment. Moreover, the expression of insulin-like growth factor-binding protein 5 (IGFBP5) was upregulated notably in the peripheral blood of GDM patients, and IGFBP5 appears to represent a direct miR-193b target. Note that silencing IGFBP5 blocked autophagy and apoptosis in HG-treated HTR8 cells, an effect that was diminished by inhibiting miR-193b. CONCLUSION: Our data indicate that aberrantly low expression of miR-193b in HG-induced trophoblasts results in massive apoptosis events by upregulating IGFBP5-induced autophagy, which may trigger GDM. Therefore, miR-193b may became a potential target for GDM therapy.

The Multi-Faced Role of PAPP-A in Post-Partum Breast Cancer: IGF-Signaling is Only the Beginning.

Insulin-like growth factor (IGF) signaling and control of local bioavailability of free IGF by the IGF binding proteins (IGFBP) are important regulators of both mammary development and breast cancer. A recent genome-wide association study (GWAS) identified small nucleotide polymorphisms that reduce the expression of IGFBP-5 as a risk factor of developing breast cancer. This observation suggests that genetic alterations leading to a decreased level of IGFBP-5 may also contribute to breast cancer. In the current review, we focus on Pregnancy-Associated Plasma Protein A (PAPP-A), a protease involved in the degradation of IGFBP-5. PAPP-A is overexpressed in the majority of breast cancers but its role in cancer has only begun to be explored. More specifically, this review aims at highlighting the role of post-partum involution in the oncogenic function of PAPP-A. Notably, we summarize recent studies indicating that PAPP-A plays a role not only in the degradation of IGFBP-5 but also in the deposition of collagen and activation of the collagen receptor discoidin 2 (DDR2) during post-partum involution. Finally, considering the immunosuppressive microenvironment of post-partum involution, we also discuss the unexpected finding made in Ewing Sarcoma that PAPP-A plays a role in immune evasion. While the immunosuppressive role of PAPP-A in breast cancer remains to be determined, collectively these studies highlight the multifaced role of PAPP-A in cancer that extends well beyond its effect on IGF-signaling.

Prolonged treatment with Y-27632 promotes the senescence of primary human dermal fibroblasts by increasing the expression of IGFBP-5 and transforming them into a CAF-like phenotype.

The Rho-kinases (ROCK) inhibitor Y-27632 has been shown to promote the growth of epidermal cells, however, its potential effects on human dermal fibroblasts (HDFs) need to be clarified. Here we show that prolonged treatment of HDFs with Y-27632 decreased their growth by inducing senescence, which was associated with induction of the senescence markers p16 and p21, and downmodulation of the ERK pathways. The senescent HDFs induced by Y-27632 acquired a cancer-associated-fibroblast (CAF)-like phenotype to promote squamous cell carcinoma (SCC) cell growth in vitro. Expression of a newly identified target of Y-27632 by RNA-seq, insulin growth factor binding protein 5 (IGFBP-5), was dramatically increased after 24 h of treatment with Y-27632. Adding recombinant IGFBP-5 protein to the culture medium produced similar phenotypes of HDFs as did treatment with Y-27632, and knockdown of IGFBP-5 blocked the Y-27632-induced senescence. Furthermore, Y-27632 induced the expression of an IGFBP-5 upstream gene, GATA4, and knockdown of GATA4 also reduced the Y-27632-induced senescence. In summary, these results demonstrate for the first time that Y-27632 promotes cellular senescence in primary HDFs by inducing the expression of IGFBP-5 and that prolonged treatment with Y-27632 potentially transforms primary HDFs into CAF-like cells.

Relationships between Breast Feeding and Breast Cancer Subtypes: Lessons Learned from Studies in Humans and in Mice.

There are differential risk relationships between parity and breast cancer according to estrogen receptor (ER) status, with an increased risk of ER- disease reduced by breastfeeding. This may be particularly relevant for understanding the higher incidence of ER- tumors in Black women, who are more likely to be parous and less likely to breastfeed than other U.S. groups. Potential mechanisms for these relationships may include effects of disordered breast involution on inflammatory milieu in the breast as well as epigenetic reprogramming in the mammary gland, which can affect cell fate decisions in progenitor cell pools. In normal breast tissue, parity has been associated with hypermethylation of FOXA1, a pioneer transcription factor that promotes the luminal phenotype in luminal progenitors, while repressing the basal phenotype. In breast tumors, relationships between FOXA1 methylation and parity were strongest among women who did not breastfeed. Here, we summarize the epidemiologic literature regarding parity, breastfeeding, and breast cancer subtypes, and review potential mechanisms whereby these factors may influence breast carcinogenesis, with a focus on effects on progenitor cell pools in the mammary gland.

IRE1α and IGF signaling predict resistance to an endoplasmic reticulum stress-inducing drug in glioblastoma cells.

To date current therapies of glioblastoma multiforme (GBM) are largely ineffective. The induction of apoptosis by an unresolvable unfolded protein response (UPR) represents a potential new therapeutic strategy. Here we tested 12ADT, a sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, on a panel of unselected patient-derived neurosphere-forming cells and found that GBM cells can be distinguished into "responder" and "non-responder". By RNASeq analysis we found that the non-responder phenotype is significantly linked with the expression of UPR genes, and in particular ERN1 (IRE1) and ATF4. We also identified two additional genes selectively overexpressed among non-responders, IGFBP3 and IGFBP5. CRISPR-mediated deletion of the ERN1, IGFBP3, IGFBP5 signature genes in the U251 human GBM cell line increased responsiveness to 12ADT. Remarkably, >65% of GBM cases in The Cancer Genome Atlas express the non-responder (ERN1, IGFBP3, IGFBP5) gene signature. Thus, elevated levels of IRE1α and IGFBPs predict a poor response to drugs inducing unresolvable UPR and possibly other forms of chemotherapy helping in a better stratification GBM patients.

The metalloproteinase Papp-aa controls epithelial cell quiescence-proliferation transition.

Human patients carrying PAPP-A2 inactivating mutations have low bone mineral density. The underlying mechanisms for this reduced calcification are poorly understood. Using a zebrafish model, we report that Papp-aa regulates bone calcification by promoting Ca2+-transporting epithelial cell (ionocyte) quiescence-proliferation transition. Ionocytes, which are normally quiescent, re-enter the cell cycle under low [Ca2+] stress. Genetic deletion of Papp-aa, but not the closely related Papp-ab, abolished ionocyte proliferation and reduced calcified bone mass. Loss of Papp-aa expression or activity resulted in diminished IGF1 receptor-Akt-Tor signaling in ionocytes. Under low Ca2+ stress, Papp-aa cleaved Igfbp5a. Under normal conditions, however, Papp-aa proteinase activity was suppressed and IGFs were sequestered in the IGF/Igfbp complex. Pharmacological disruption of the IGF/Igfbp complex or adding free IGF1 activated IGF signaling and promoted ionocyte proliferation. These findings suggest that Papp-aa-mediated local Igfbp5a cleavage functions as a [Ca2+]-regulated molecular switch linking IGF signaling to bone calcification by stimulating epithelial cell quiescence-proliferation transition under low Ca2+ stress.

MicroRNA-325-3p prevents sevoflurane-induced learning and memory impairment by inhibiting Nupr1 and C/EBPß/IGFBP5 signaling in rats.

Endoplasmic reticulum stress-induced neuronal apoptosis contributes to neurotoxicity observed after sevoflurane exposure. However, the molecular mechanism underlying the resulting learning and memory impairments remains unknown. Here, we investigated the roles of miR-325-3p and Nupr1 in sevoflurane-induced learning and memory impairments in neonatal rats and HCN-2 human cortical neuronal cells. We found that in both neonatal rats and HCN-2 cells, sevoflurane exposure impairs learning and memory in neonatal rats and increases expression of Nupr1, the endoplasmic reticulum stress marker proteins C/EBPß and IGFBP5, and the apoptosis-related protein markers cleaved-Caspase-3 and Bax. Using bioinformatics tools to identify microRNAs that bind to Nupr1, we found that miR-325-3p is downregulated in hippocampal neurons exposed to sevoflurane. Moreover, Nupr1 knockdown and miR-325-3p overexpression improved the rats' performance in learning and memory tests and reduced sevoflurane-induced apoptosis in vitro and in vivo. These results suggest that miR-325-3p blocks sevoflurane-induced learning and memory impairments by inhibiting Nupr1 and the downstream C/EBPß/IGFBP5 signaling axis in neonatal rats. MiR-325-3p may therefore be a useful therapeutic target in sevoflurane-induced neurotoxicity.

Temporal Expression and Cellular Localization of PAPPA2 in the Developing Kidney of Rat.

PAPPA2 is a metalloproteinase which cleaves insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5, and its role in pregnancy and postnatal growth is primarily studied. Using exclusion mapping, we reported a subcongenic (26-P) rat where a 0.71-Mbp region containing the pregnancy-associated plasma protein a2 (Pappa2) allele of salt-insensitive Brown Norway (BN) was introgressed into Dahl saltsensitive (SS) genetic background, resulting in the reduction of salt sensitivity. Pappa2 was differentially expressed in the adult kidney of 26-P and SS rats. Here, the expression and cellular localization of Pappa2 in embryonic and postnatal kidneys of 26-P and SS rats were examined. Pappa2 mRNA expression was 5-fold higher in the embryonic kidney (day 20.5) of the 26-P rat compared with the SS rat. Pappa2 mRNA expression progressively increased with the development of kidney, reaching a peak at postnatal day 5 before trending downward in subsequent stages of development in both strains. At all tested time points, Pappa2 remained higher in the 26-P compared with the SS rat kidney. Immunohistochemistry studies localized PAPPA2 in the ureteric bud (UB) and distal part of S-shaped body. PAPPA2 was colocalized with IGFBP-5 in the UB and Na+/K+/2Cl- cotransporter-stained tubules, respectively. Future studies are needed to determine the role of Pappa2 in kidney development and mechanistic pathways involved in this process.

HASPIN is involved in the progression of gallbladder carcinoma.

BACKGROUND: Gallbladder carcinoma (GBC) is a common malignant tumor of the biliary system, but the current treatment of GBC is unsatisfactory. Therefore, new treatment targets and strategies are urgently needed. METHODS: The expression of HASPIN in GBC was detected by immunohistochemical staining. HASPIN knockdown cell model was constructed by lentivirus infection, and the infection efficiency of lentivirus and knockdown efficiency of shHASPIN were verified by fluorescence immunoassay, qRT-PCR and Western blot. The effects of HASPIN knockdown on cell proliferation, clone-formation ability and apoptosis were determined by MTT, clone formation assay, flow cytometry and Human Apoptosis Antibody Array in vitro. Besides, the effect of HASPIN knockdown on the growth of GBC solid tumors was demonstrated in vivo. RESULTS: The expression of HASPIN in GBC was up-regulated and positively correlated with the pathological grade of GBC. ShHASPIN significantly down-regulated the mRNA and protein levels of HASPIN, suggesting that HASPIN knockdown cell model was successfully constructed in vitro. After HASPIN knockdown, the proliferation and clone-formation ability of GBC cells were observably inhibited, the apoptotic levels were markedly increased, and the expression of Caspase 3, IGFBP-5, p21 and sTNF-R1 related to apoptotic pathway was up-regulated. Furthermore, HASPIN knockdown inhibited the growth of GBC in vivo. CONCLUSION: HASPIN was up-regulated in GBC and played an important role in promoting the progress of GBC.