DDX20 is required for cell-cycle reentry of prospermatogonia and establishment of spermatogonial stem cell pool during testicular development in mice.

RNA-binding proteins (RBPs), as key regulators of mRNA fate, are abundantly expressed in the testis. However, RBPs associated with human male infertility remain largely unknown. Through bioinformatic analyses, we identified 62 such RBPs, including an evolutionarily conserved RBP, DEAD-box helicase 20 (DDX20). Male germ-cell-specific inactivation of Ddx20 at E15.5 caused T1-propsermatogonia (T1-ProSG) to fail to reenter cell cycle during the first week of testicular development in mice. Consequently, neither the foundational spermatogonial stem cell (SSC) pool nor progenitor spermatogonia were ever formed in the knockout testes. Mechanistically, DDX20 functions to control the translation of its target mRNAs, many of which encode cell-cycle-related regulators, by interacting with key components of the translational machinery in prospermatogonia. Our data demonstrate a previously unreported function of DDX20 as a translational regulator of critical cell-cycle-related genes, which is essential for cell-cycle reentry of T1-ProSG and formation of the SSC pool.

Decoding the spermatogonial stem cell niche under physiological and recovery conditions in adult mice and humans.

The intricate interaction between spermatogonial stem cell (SSC) and testicular niche is essential for maintaining SSC homeostasis; however, this interaction remains largely uncharacterized. In this study, to characterize the underlying signaling pathways and related paracrine factors, we delineated the intercellular interactions between SSC and niche cell in both adult mice and humans under physiological conditions and dissected the niche-derived regulation of SSC maintenance under recovery conditions, thus uncovering the essential role of C-C motif chemokine ligand 24 and insulin-like growth factor binding protein 7 in SSC maintenance. We also established the clinical relevance of specific paracrine factors in human fertility. Collectively, our work on decoding the adult SSC niche serves as a valuable reference for future studies on the aetiology, diagnosis, and treatment of male infertility.

Identification of quiescent FOXC2+ spermatogonial stem cells in adult mammals.

In adult mammals, spermatogenesis embodies the complex developmental process from spermatogonial stem cells (SSCs) to spermatozoa. At the top of this developmental hierarchy lie a series of SSC subpopulations. Their individual identities as well as the relationships with each other, however, remain largely elusive. Using single-cell analysis and lineage tracing, we discovered both in mice and humans the quiescent adult SSC subpopulation marked specifically by forkhead box protein C2 (FOXC2). All spermatogenic progenies can be derived from FOXC2+ SSCs and the ablation of FOXC2+ SSCs led to the depletion of the undifferentiated spermatogonia pool. During germline regeneration, FOXC2+ SSCs were activated and able to completely restore the process. Germ cell-specific Foxc2 knockout resulted in an accelerated exhaustion of SSCs and eventually led to male infertility. Furthermore, FOXC2 prompts the expressions of negative regulators of cell cycle thereby ensures the SSCs reside in quiescence. Thus, this work proposes that the quiescent FOXC2+ SSCs are essential for maintaining the homeostasis and regeneration of spermatogenesis in adult mammals.

YWK-II/APLP2 inhibits TGF-ß signaling by interfering with the TGFBR2-Hsp90 interaction.

Transforming growth factor-ß (TGF-ß) regulates multiple cellular biological processes by activating TGF-ß type I receptors (TGFBR1) and type II receptors (TGFBR2), and Hsp90 stabilizes these receptors through specific interactions. In many malignancies, one of the most deregulated signaling pathways is the TGF-ß signaling pathway, which is often inactivated by mutations or deregulation of TGF-ß type II receptors (TGFBR2). However, the molecular mechanisms are not well understood. In this study, we show that YWK-II/APLP2, an immediately early response gene for TGF-ß signaling, inhibits TGF-ß signaling by promoting the degradation of the TGFBR2 protein. Knockdown of YWK-II/APLP2 increases the TGFBR2 protein level and sensitizes cells to TGF-ß stimulation, while YWK-II/APLP2 overexpression destabilizes TGFBR2 and desensitizes cells to TGF-ß. Mechanistically, YWK-II/APLP2 is associated with TGFBR2 in a TGF-ß activity-dependent manner, binds to Hsp90 to interfere with the interaction between TGFBR2 and Hsp90, and leads to enhanced ubiquitination and degradation of TGFBR2. Taken together, YWK-II/APLP2 is involved in negatively regulating the duration and intensity of TGF-ß/Smad signaling and suggests that aberrantly high expression of YWK-II/APLP2 in malignancies may antagonize the growth inhibition mediated by TGF-ß signaling and play a role in carcinogenesis.

CHD4 acts as a critical regulator in the survival of spermatogonial stem cells in mice†.

Spermatogenesis is sustained by homeostatic balance between the self-renewal and differentiation of spermatogonial stem cells, which is dependent on the strict regulation of transcription factor and chromatin modulator gene expression. Chromodomain helicase DNA-binding protein 4 is highly expressed in spermatogonial stem cells but roles in mouse spermatogenesis are not fully understood. Here, we report that the germ-cell-specific deletion of chromodomain helicase DNA-binding protein 4 resulted in complete infertility in male mice, with rapid loss of spermatogonial stem cells and excessive germ cell apoptosis. Chromodomain helicase DNA-binding protein 4-knockdown in cultured spermatogonial stem cells also promoted the expression of apoptosis-related genes and thereby activated the tumor necrosis factor signaling pathway. Mechanistically, chromodomain helicase DNA-binding protein 4 occupies the genomic regulatory region of key apoptosis-related genes, including Jun and Nfkb1. Together, our findings reveal the determinant role of chromodomain helicase DNA-binding protein 4 in spermatogonial stem cells survival in vivo, which will offer insight into the pathogenesis of male sterility and potential novel therapeutic targets.

RING finger 138 deregulation distorts NF-кB signaling and facilities colitis switch to aggressive malignancy.

Prolonged activation of nuclear factor (NF)-кB signaling significantly contributes to the development of colorectal cancer (CRC). New therapeutic opportunities are emerging from targeting this distorted cell signaling transduction. Here, we discovered the critical role of RING finger 138 (RNF138) in CRC tumorigenesis through regulating the NF-кB signaling, which is independent of its Ubiquitin-E3 ligase activity involved in DNA damage response. RNF138-/- mice were hyper-susceptible to the switch from colitis to aggressive malignancy, which coincided with sustained aberrant NF-кB signaling in the colonic cells. Furthermore, RNF138 suppresses the activation of NF-кB signaling pathway through preventing the translocation of NIK and IKK-Beta Binding Protein (NIBP) to the cytoplasm, which requires the ubiquitin interaction motif (UIM) domain. More importantly, we uncovered a significant correlation between poor prognosis and the downregulation of RNF138 associated with reinforced NF-кB signaling in clinical settings, raising the possibility of RNF138 dysregulation as an indicator for the therapeutic intervention targeting NF-кB signaling. Using the xenograft models built upon either RNF138-dificient CRC cells or the cells derived from the RNF138-dysregulated CRC patients, we demonstrated that the inhibition of NF-кB signaling effectively hampered tumor growth. Overall, our work defined the pathogenic role of aberrant NF-кB signaling due to RNF138 downregulation in the cascade events from the colitis switch to colonic neoplastic transformation and progression, and also highlights the possibility of targeting the NF-кB signaling in treating specific subtypes of CRC indicated by RNF138-ablation.

Progress on spermatogonial stem cell microenvironment.

Spermatogonial stem cells (SSCs) are germ cells (GCs) with long-term self-renewal and differentiation potential in testis, namely tissue stem cells located on the basement membrane, whose self-renewal and differentiation are regulated by the surrounding microenvironment. In recent years, the research of SSCs has made a series of important progress, which brings the hope for the clinical treatment of some male infertility patients. Among them, the microenvironment is particularly important in regulating SSCs. The microenvironment is responsible for integrating the effects of different types of cell components, extracellular matrix, extracellular regulatory molecules and hormones on SSCs, thus regulating the fate of SSCs. The research on SSCs microenvironment has gradually become one of the main contents of stem cell research. In this review, we mainly summarize the cell composition, regulatory factors and characteristics of mouse SSCs microenvironment, thereby providing background information for in-depth study on the structure and function of SSCs microenvironment, and opportunity to find more abundant cell phenotypes and microenvironmental factors through multiple research models in the future.

The phosphorylation of the Smad2/3 linker region by nemo-like kinase regulates TGF-ß signaling.

Smad2 and Smad3 (Smad2/3) are structurally similar proteins that primarily mediate the transforming growth factor-ß (TGF-ß) signaling responsible for driving cell proliferation, differentiation, and migration. The dynamics of the Smad2/3 phosphorylation provide the key mechanism for regulating the TGF-ß signaling pathway, but the details surrounding this phosphorylation remain unclear. Here, using in vitro kinase assay coupled with mass spectrometry, we identified for the first time that nemo-like kinase (NLK) regulates TGF-ß signaling via modulation of Smad2/3 phosphorylation in the linker region. TGF-ß-mediated transcriptional and cellular responses are suppressed by NLK overexpression, whereas NLK depletion exerts opposite effects. Specifically, we discovered that NLK associates with Smad3 and phosphorylates the designated serine residues located in the linker region of Smad2 and Smad3, which inhibits phosphorylation at the C terminus, thereby decreasing the duration of TGF-ß signaling. Overall, this work demonstrates that phosphorylation on the linker region of Smad2/3 by NLK counteracts the canonical phosphorylation in response to TGF-ß signals, thus providing new insight into the mechanisms governing TGF-ß signaling transduction.

Panoramic transcriptome analysis and functional screening of long noncoding RNAs in mouse spermatogenesis.

Long noncoding RNAs (lncRNAs) have emerged as diverse functional regulators involved in mammalian development; however, large-scale functional investigation of lncRNAs in mammalian spermatogenesis in vivo is lacking. Here, we delineated the global lncRNA expression landscape in mouse spermatogenesis and identified 968 germ cell signature lncRNAs. By combining bioinformatics and functional screening, we identified three functional lncRNAs (Gm4665, 1700027A15Rik, and 1700052I22Rik) that directly influence spermatogenesis in vivo. Knocking down Gm4665 hampered the development of round spermatids into elongating spermatids and disrupted key spermatogenic gene expression. Mechanistically, lncRNA Gm4665 localized in the nucleus of round spermatids and occupied the genomic regulatory region of important spermatogenic genes including Ip6k1 and Akap3 These findings provide a valuable resource and framework for future functional analysis of lncRNAs in spermatogenesis and their potential roles in other biological processes.

PDCD6 cooperates with C-Raf to facilitate colorectal cancer progression via Raf/MEK/ERK activation.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies, and it's expected that the CRC burden will substantially increase in the next two decades. New biomarkers for targeted treatment and associated molecular mechanism of tumorigenesis remain to be explored. In this study, we investigated whether PDCD6 plays an oncogenic role in colorectal cancer and its underlying mechanism. METHODS: Programmed cell death protein 6 (PDCD6) expression in CRC samples were analyzed by immunohistochemistry and immunofluorescence. The prognosis between PDCD6 and clinical features were analyzed. The roles of PDCD6 in cellular proliferation and tumor growth were measured by using CCK8, colony formation, and tumor xenograft in nude mice. RNA-sequence (RNA-seq), Mass Spectrum (MS), Co-Immunoprecipitation (Co-IP) and Western blot were utilized to investigate the mechanism of tumor progression. Immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) were performed to determine the correlation of PDCD6 and MAPK pathway. RESULTS: Higher expression levels of PDCD6 in tumor tissues were associated with a poorer prognosis in patients with CRC. Furthermore, PDCD6 increased cell proliferation in vitro and tumor growth in vivo. Mechanistically, RNA-seq showed that PDCD6 could affect the activation of the MAPK signaling pathway. PDCD6 interacted with c-Raf, resulting in the activation of downstream c-Raf/MEK/ERK pathway and the upregulation of core cell proliferation genes such as MYC and JUN. CONCLUSIONS: These findings reveal the oncogenic effect of PDCD6 in CRC by activating c-Raf/MEK/ERK pathway and indicate that PDCD6 might be a potential prognostic indicator and therapeutic target for patients with colorectal cancer.

A long noncoding RNA binding to QKI-5 regulates germ cell apoptosis via p38 MAPK signaling pathway.

Spermatogenesis is the complex process of male germline development and requires coordinated interactions by multiple gene products that undergo strict developmental regulations. Increasing evidence has suggested that a number of long noncoding RNAs (lncRNAs) may function as important regulatory molecules in various physiological and pathological processes by binding to specific proteins. Here, we identified a subset of QKI-5-binding lncRNAs in the mouse testis through the integrated analyses of RNA immunoprecipitation (RIP)-microarray and biological verification. Among the lncRNAs, we revealed that NONMMUT074098.2 (Lnc10), which was highly expressed in the spermatogonia and spermatocytes of the testis, interacted with QKI-5. Furthermore, Lnc10 depletion promoted germ cell apoptosis via the activation of p38 MAPK, whereas the simultaneous knockdown of QKI-5 could rescue the apoptotic phenotype and the activation of p38 MAPK, which were induced by the loss of Lnc10. These data indicated that the Lnc10-QKI-5 interaction was associated with the regulatory roles of QKI-5 and that the Lnc10-QKI-5 interaction inhibited the regulation of QKI-5 on the downstream p38 MAPK signaling pathway. Additionally, we functionally characterized the biological roles of Lnc10 and found that the knockdown of Lnc10 promoted the apoptosis of spermatogenic cells in vivo; this suggested that Lnc10 had an important biological role in mouse spermatogenesis. Thus, our study provides a potential strategy to investigate the biological significance of lncRNA-RBP interactions during male germline development.

P53-R273H mutation enhances colorectal cancer stemness through regulating specific lncRNAs.

BACKGROUND: TP53 is one of the most frequently mutated genes among all cancer types, and TP53 mutants occur more than 60% in colorectal cancer (CRC). Among all mutants, there are three hot spots, including p53-R175H, p53-R248W and p53-R273H. Emerging evidence attributes cancer carcinogenesis to cancer stem cells (CSCs). Long noncoding RNAs (lncRNAs) play crucial roles in maintaining the stemness of CSCs. However, it is unknown if mutant p53-regulated lncRNAs are implicated in the maintenance of CSC stemness. METHODS: RNA-sequencing (RNA-seq) and ChIP-sequencing (ChIP-seq) were used to trace the lncRNA network regulated by p53-R273H in HCT116 endogenous p53 point mutant spheroid cells generated by the somatic cell knock-in method. RT-qPCR was used to detect lncRNA expression patterns, verifying the bioinformatics analysis. Transwell, spheroid formation, fluorescence activated cell sorter (FACS), xenograft nude mouse model, tumor frequency assessed by extreme limiting dilution analysis (ELDA), Western blot assays and chemoresistance analysis were performed to elucidate the functions and possible mechanism of lnc273-31 and lnc273-34 in cancer stem cells. RESULTS: p53-R273H exhibited more characteristics of CSC than p53-R175H and p53-R248W. RNA-seq profiling identified 37 up regulated and 4 down regulated differentially expressed lncRNAs regulated by p53-R273H. Combined with ChIP-seq profiling, we further verified two lncRNAs, named as lnc273-31 and lnc273-34, were essential in the maintenance of CSC stemness. Further investigation illustrated that lnc273-31 or lnc273-34 depletion dramatically diminished colorectal cancer migration, invasion, cancer stem cell self-renewal and chemoresistance in vitro. Moreover, the absence of lnc273-31 or lnc273-34 dramatically delayed cancer initiation and tumorigenic cell frequency in vivo. Also, lnc273-31 and lnc273-34 have an impact on epithelial-to mesenchymal transition (EMT). Finally, lnc273-31 and lnc273-34 were significantly highly expressed in CRC tissues with p53-R273H mutation compared to those with wildtype p53. CONCLUSIONS: The present study unveiled a high-confidence set of lncRNAs regulated by p53-R273H specific in colorectal CSCs. Furthermore, we demonstrated that two of them, lnc273-31 and lnc273-34, were required for colorectal CSC self-renewal, tumor propagation and chemoresistance. Also, the expression of these two lncRNAs augmented in colorectal cancer patient samples with p53-R273H mutation. These two lncRNAs may serve as promising predictors for patients with p53-R273H mutation and are vital for chemotherapy.

Rhomboid domain-containing protein 1 promotes breast cancer progression by regulating the p-Akt and CDK2 levels.

BACKGROUND: Our previous work revealed that rhomboid domain-containing protein 1 (RHBDD1) participates in the modulation of cell growth and apoptosis in colorectal cancer cells. This study aimed to investigate the function of RHBDD1 in regulating breast cancer progression and its underlying molecular basis. METHODS: Immunohistochemistry was performed to evaluate RHBDD1 expression in 116 breast cancer tissue and 39 adjacent normal tissue and expression of RHBDD1, phospho-Akt (p-Akt) and cyclin-dependent kinase 2 (CDK2) in the same 84 breast cancer specimens. RHBDD1-knock-out cells were established using breast cancer cell lines. In vitro studies were carried out to estimate the function of RHBDD1 in cell proliferation, migration and invasion. Fluorescence microscopy assay and flow cytometric analysis were used to measure apoptosis and cell cycle regulation. RNA sequencing and western blot analysis were used to investigate the molecular mechanisms of RHBDD1. RESULTS: RHBDD1 was highly up-regulated in breast cancer tissue compared with that in normal tissue and associated with pathological tumor (pT) stage, pathological tumor-node-metastasis (pTNM) stage and estrogen receptor (ER) expression. RHBDD1 up-regulation was associated with poor prognosis in several subtypes of breast cancer. Deletion of RHBDD1 promoted apoptosis and suppressed proliferation, migration and invasion in breast cancer cells. RHBDD1 deletion suppressed Akt activation and decreased CDK2 protein level via proteasome pathway, thus inhibited cell cycle progression and G1/S phase transition. Moreover, the protein level of RHBDD1, p-Akt and CDK2 was significantly positively correlated in breast cancer tissue. CONCLUSIONS: Our study reveals that RHBDD1 promotes breast cancer progression by regulating p-Akt and CDK2 protein levels, and might be a potential biomarker and prognostic indicator for breast cancer patients.

RNF138 confers cisplatin resistance in gastric cancer cells via activating Chk1 signaling pathway.

Chemotherapy resistance represents a major issue associated with gastric cancer (GC) treatment, and arises through multiple mechanisms, including modulation of the cell-cycle check point. Several ubiquitin kinases, including RING finger protein 138 (RNF138), have been reported to mediate the G2/M phase arrest. In this study, we investigated the role of RNF138 in the development of cisplatin resistance of two GC cell lines. We show that RNF138 levels are higher in cisplatin-resistant cell lines, compared with cisplatin-sensitive cells, and RNF138 expression was elevated during drug withdrawal following the cisplatin treatment. Using gene overexpression and silencing, we analyzed the impact of altering RNF138 level on GC cell viability, apoptosis, and cell cycle phenotypes in two isogenic cisplatin-sensitive and resistant cell lines. We show that RNF138 overexpression increased GC cell viability, decreased apoptosis and delayed cell cycle progression in the cisplatin-sensitive GC cells. Conversely, RNF138 silencing produced opposite phenotypes in the cisplatin-resistant cells. Moreover, RNF138-dependent phosphorylation of Chk1 was seen in GC cells, indicating a novel connection between cisplatin-induced DNA damage and apoptosis. Collectively, these data suggest that RNF138 modulates the cisplatin resistance in the GC cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, RNF138 can also be used as a marker to monitor the development of cisplatin resistance in GC treatment.

Correction to: RHBDD1 promotes colorectal cancer metastasis through the Wnt signaling pathway and its downstream target ZEB1.

In the publication of this article [1], there is an error in the Methods section at paragraph Cell culture and reagents and the Additional file2: Fig. S1 was erroneous linked.

RHBDD1 promotes colorectal cancer metastasis through the Wnt signaling pathway and its downstream target ZEB1.

BACKGROUND: 40-50% of colorectal cancer (CRC) patients develop metastatic disease; the presence of metastasis hinders the effective treatment of cancer through surgery, chemotherapy and radiotherapy, which makes 5-year survival rate extremely low; therefore, studying CRC metastasis is crucial for disease therapy. In the present study, we investigated the role of rhomboid domain containing 1 (RHBDD1) in tumor metastasis of CRC. METHODS: The expression of RHBDD1 was analyzed in 539 colorectal tumor tissues for its correlation with lymphatic metastasis and distal metastasis. Transwell assay in vitro and pleural metastasis analysis in vivo were performed to determine the functions of RHBDD1 during CRC cells metastasis. RNA-seq analysis, TOP/FOP flash reporter assay, western blot and transwell assay were performed to investigate the underlying mechanism for the function of RHBDD1 on Wnt signaling pathway. Bioinformatics analysis was conducted to investigate epithelial-mesenchymal transition (EMT) and stemness in HCT-116 cells. Tissue microarray analysis, Q-PCR and western blot were performed to determine the correlation of RHBDD1 and Zinc Finger E-Box Binding Homeobox 1 (ZEB1). RESULTS: In this study, we found that RHBDD1 expression was positively correlated with lymphatic metastasis and distal metastasis in 539 colorectal tumor tissues. RHBDD1 expression can promote CRC cells metastasis in vitro and in vivo. RNA-Seq analysis showed that the Wnt signaling pathway played a key role in this metastatic regulation. RHBDD1 mainly regulated ser552 and ser675 phosphorylation of ß-catenin to activate the Wnt signaling pathway. Rescuing ser552 and ser675 phosphorylation of ß-catenin resulted in the recovery of signaling pathway activity, migration, and invasion in CRC cells. RHBDD1 promoted EMT and a stem-like phenotype of CRC cells. RHBDD1 regulated the Wnt/ß-catenin target gene ZEB1, a potent EMT activator, at the RNA and protein levels. Clinically, RHBDD1 expression was positively correlated with ZEB1 at the protein level in 71 colon tumor tissues. CONCLUSIONS: Our findings therefore indicated that RHBDD1 can promote CRC metastasis through the Wnt signaling pathway and ZEB1. RHBDD1 may become a new therapeutic target or clinical biomarker for metastatic CRC.

Rnf138 deficiency promotes apoptosis of spermatogonia in juvenile male mice.

Spermatogenesis, the process by which haploid sperm cells are produced from a diploid precursor cell, is essential for sexual reproduction. Here, we report that RING-finger protein 138 (Rnf138) is highly expressed in testes, especially in spermatogonia and spermatocytes. The role of Rnf138 in spermatogenesis was examined using a Rnf138-knockout mouse model. Rnf138 deficiency resulted in increased apoptosis in spermatogenic cells, loss of proliferative spermatogonia, delayed development of spermatozoa and impaired fertility. The proportion of PLZF+Ki67+ cells within the PLZF+ population decreased in the knockout mice. The phenotype was further assessed by RNA-sequencing (RNA-seq), which determined that the expression levels of many genes involved in spermatogenesis were altered in the testis of Rnf138-knockout mice. Thus, Rnf138 deficiency promotes the apoptosis of spermatogenic cells, which may have been caused by the aberrant proliferation of spermatogonia in mouse testis development.

RHBDD1 upregulates EGFR via the AP-1 pathway in colorectal cancer.

Our previous study showed that RHBDD1 can activate the EGFR signaling pathway to promote colorectal cancer growth. In the present study, EGFR was decreased when RHBDD1 was knocked down or inactivated. Further analysis found that c-Jun and EGFR protein expression was decreased in RHBDD1 knockdown and inactivated cells. c-Jun overexpression in RHBDD1-inactivated cells rescued EGFR expression in a dose-dependent manner. RHBDD1 overexpression in RHBDD1-inactivated cells restored EGFR expression, but this effect was counteracted by c-Jun knockdown. Furthermore, EGFR and c-Jun were attenuated in the RHBDD1 knockdown and inactivated groups in animal tumor models. Tissue microarray assays demonstrated a correlation between RHBDD1 and EGFR in colorectal cancer patients. Therefore, our findings indicate that RHBDD1 stimulates EGFR expression by promoting the AP-1 pathway.

Role of perivascular adipose tissue in nicotine­induced endothelial cell inflammatory responses.

Smoking is considered to be one of the primary causes of atherosclerosis and vascular injury. Previous studies have shown that nicotine in tobacco can lead to vascular inflammation and endothelial dysfunction. Perivascular adipose tissue (PVAT) is known to secrete various types of adipokines to maintain vascular homeostasis. The present study investigated whether nicotine­induced PVAT malfunction can accelerate endothelial inflammation and eventually lead to endothelial dysfunction. The levels of inflammatory adipokines, including nuclear factor (NF)­κB, interleukin (IL)­1ß, IL­6 and tumor necrosis factor (TNF)­α, the ICAM­1 and VCAM­1 adhesion molecules and secretion of adiponectin were assessed in mature adipocytes and endothelial cells cultured alone or in co­culture under nicotine stimulation. It was found that nicotine reduced the secretion of adiponectin and stimulated secretion of the NF­κB, IL­1ß, IL­6 and TNF­α inflammatory adipokines in mature adipocytes. Although nicotine stimulated endothelial cells to secrete IL­1ß and IL­6, no significant increase in the secretion of TNF­α was observed. The co­culture of mature adipocytes with endothelial cells markedly augmented the expression of the NF­κB, IL­1ß, IL­6 and TNF­α inflammatory adipokines and the ICAM­1 and VCAM­1 adhesion molecules, and significantly lowered the levels of adiponectin. These findings suggested that nicotine induced mature adipocyte dysfunction, which caused the abnormal secretion of adiponectin and inflammatory adipokines, and exacerbated endothelial inflammation. These findings also suggested a mechanism whereby nicotine induced the secretion of adiponectin and inflammatory cytokines by adipocytes. The results of the present study elucidated a novel pathway induced by cigarette smoke, which contributed to atherosclerosis and vascular injury.