Opposing actions of the progesterone metabolites, 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) on mitosis,apoptosis, and expression of Bcl-2, Bax and p21 in human breast cell lines

Previous studies have shown that breast tissues and breast cell lines convert progesterone (P) to 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) and that 3αHP suppresses, whereas 5αP promotes, cell proliferation and detachment. The objectives of the current studies were to determine if the 5αP- and 3αHP-induced changes in cell numbers are due to altered rates of mitosis and/or apoptosis, and if 3αHP and 5αP act on tumorigenic and non-tumorigenic cells, regardless of estrogen (E) and P receptor status. The studies were conducted on tumorigenic (MCF-7, MDA-MB-231, T47D) and non-tumorigenic (MCF-10A) human breast cell lines, employing several methods to assess the effects of the hormones on cell proliferation, mitosis, apoptosis and expression of Bcl-2, Bax and p21. In all four cell lines, 5αP increased, whereas 3αHP decreased cell numbers, [³H]thymidine uptake and mitotic index. Apoptosis was stimulated by 3αHP and suppressed by 5αP. 5αP resulted in increases in Bcl-2/Bax ratio, indicating decreased apoptosis; 3αHP resulted in decreases in Bcl-2/Bax ratio, indicating increased apoptosis. The effects of either 3αHP or 5αP on cell numbers, [³H]thymidine uptake, mitosis, apoptosis, and Bcl-2/Bax ratio, were abrogated when cells were treated simultaneously with both hormones. The expression of p21 was increased by 3αHP, and was unaffected by 5αP. The results provide the first evidence that 5αP stimulates mitosis and suppresses apoptosis, whereas 3αHP inhibits mitosis and stimulates apoptosis. The opposing effects of 5αP and 3αHP were observed in all four breast cell lines examined and the data suggest that all breast cancers (estrogen-responsive and unresponsive) might be suppressed by blocking 5αP formation and/or increasing 3αHP. The findings further support the hypothesis that progesterone metabolites are key regulatory hormones and that changes in their relative concentrations in the breast microenvironment determine whether breast tissues remain normal or become cancerous.     

SOCS-1 is involved in TNF-α-induced mitochondrial dysfunction and apoptosis in renal tubular epithelial cells

Tumor necrosis factor-α (TNF-α) is suggested to induce mitochondrial dysfunction and apoptosis of renal tubular epithelial cells that possibly exacerbates renal function in chronic kidney disease (CKD). Here we investigated whether suppressor of cytokine signaling-1 (SOCS-1), an inhibitor of cytokine signaling, was involved in TNF-α-induced human renal tubular epithelial cells (HKCs) oxidative stress and apoptosis. TNF-α promoted the protein and mRNA expression of SOCS-1 in a time and dose dependent manner, along with increased cell apoptosis and activation of apoptosis signal regulating kinase-1(ASK1) in HKCs. Furthermore, overexpression of SOCS-1 in HKCs reduced TNF-α-mediated oxidative stress and apoptosis. Meanwhile, We also found that overexpression of SOCS-1 could regulate the activity of JAK/STAT signaling pathway. In addition, a specific JAK2 inhibitor, AG490, that both attenuated TNF-α-induced oxidative stress, also reduced apoptosis. Taken together, overexpression of SOCS-1 prevented TNF-α-mediated cell oxidative stress and apoptosis may be via suppression of JAK/STAT signaling pathway activation in HKCs.     

Synthesis of Ethyl Tfa- and Tfp- Amino Acid-p-Aminobenzoates and Their Juvenile Hormone Test

Adipocytes can function as endocrine cells secreting a variety of adipocytokines including tumor necrosis factor (TNF)-α. Treatment of cultured mouse 3T3-L1 preadipocytes with TNF-α induced apoptosis, as was evident from increases in nuclear condensation and caspase-3 activity, but differentiated adipocytes during the maturation phase showed resistance to apoptosis by TNF-α. Antioxidants effectively reduced TNF-α-induced apoptosis in preadipocytes, indicating the involvement of reactive oxygen species. Exposure of preadipocytes to calcium ionophore A23187 reduced TNF-α-induced apoptosis, which was accompanied by increased production of prostaglandins (PGs) E₂ and PGF_2α. TNF-αpreferentially promoted gene expression of cyclooxygenase (COX)-2 without affecting that of COX-1. Consistently, NS-398, a COX-2 inhibitor, stimulated TNF-α-induced apoptosis, which was reversed by exogenous PGE₂ and PGF_2α. These results indicate that endogenous PGE₂ and PGF_2α synthesized by preadipocytes through the induction of COX-2 can serve as anti-apoptotic factors against apoptosis by TNF-α.     

Genetically recessive mutant of human monocytic leukemia U937 resistant to tumor necrosis factor-α-induced apoptosis

Tumor necrosis factor-α (TNF-α) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF-α-induced apoptosis, we isolated and characterized a novel TNF-α-resistant variant, U937/TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF-α and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF-α in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF-α-induced apoptosis signaling. In UA cells, TNF-α-induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl-2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin-1β converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death-inducing receptor and mitochondria. J. Cell. Physiol. 174:179–185, 1998. © 1998 Wiley-Liss, Inc.     

Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations in PKD1, the gene for polycystin-1 (PC1), a transmembrane protein with a cytoplasmic C-terminus that interacts with numerous signaling molecules, including Gα12. The functions of PC1 and the mechanisms of cyst development leading to renal failure are complex. Recently, we reported that PC1 expression levels modulate activity of Gα12-stimulated apoptosis (Yu et al., J. Biol. Chem. 2010 285(14):10243-51). Herein, a mutational analysis of Gα12 and PC1 was undertaken to identify regions required for their interaction and ability to modulate apoptosis. A set of Gα12 mutations with systematic replacement of six amino acids with NAAIRS was tested for binding to the PC1 C-terminus in GST pulldowns. Additionally, a series of deletions within the PC1 C-terminus was examined for binding to Gα12. We identified 3 NAAIRS substitutions in Gα12 that completely abrogated binding, and identified a previously described 74 amino acid Gαi/o binding domain in the PC1 C-terminus as necessary for Gα12 interaction. The functional consequences of uncoupling PC1/Gα12 binding were studied in apoptosis assays utilizing HEK293 cells with inducible PC1 overexpression. Gα12 mutants deficient in PC1 binding were refractory to PC1 inhibition of Gα12-stimulated apoptosis. Likewise, deletion of the Gα12-interacting sequence from the PC1 cytoplasmic domain abrogated its inhibition of Gα12-stimulated apoptosis. Based on the crystal structure of Gα12, the PC1 interaction sites are likely to reside on exposed regions within the G protein helical domain. These structural details should facilitate the design of reagents to uncouple PC1/Gα12 signaling in ADPKD.     

TNFα-induced apoptosis enabled by CCN1/CYR61: pathways of reactive oxygen species generation and cytochrome c release

Although TNFα is a strong inducer of apoptosis, its cytotoxicity in most normal cells in vitro requires blockade of NFκB signaling or inhibition of de novo protein synthesis, typically by the addition of cycloheximide. However, several members of CCN (CYR61/CTGF/NOV) family of extracellular matrix proteins enable TNFα-dependent apoptosis in vitro without inhibiting NFκB or de novo protein synthesis, and CCN1 (CYR61) is essential for optimal TNFα cytotoxicity in vivo. Previous studies showed that CCN1 unmasks the cytotoxicity of TNFα by binding integrins α(v)β(5), α(6)β(1), and the cell surface heparan sulfate proteoglycan syndecan 4 to induce the accumulation of a high level of reactive oxygen species (ROS), leading to a biphasic activation of JNK necessary for apoptosis. Here we show for the first time that CCN1 interacts with the low density lipoprotein receptor-related protein 1 (LRP1) in a protein complex, and that binding to LRP1 is critical for CCN1-induced ROS generation and apoptotic synergism with TNFα. We also found that neutral sphingomyelinase 1 (nSMase1), which contributes to CCN1-induced ROS generation, is required for CCN1/TNFα-induced apoptosis. Furthermore, CCN1 promotes the activation of p53 and p38 MAPK, which mediate enhanced cytochrome c release to amplify the cytotoxicity of TNFα. By contrast, LRP1, nSMase1, p53, and p38 MAPK are not required when TNFα-dependent apoptosis is facilitated by the presence of cycloheximide, indicating that they function in the CCN1 signaling pathway that converges with TNFα-induced signaling events. Since CCN1/CYR61 is a physiological regulator of TNFα cytotoxicity at least in some contexts, these findings may reveal important mediators of TNFα-induced apoptosis in vivo and identify potential therapeutic targets for thwarting TNFα-dependent tissue damage.     

Chemical inhibition of HSP90 inhibits TNF-α mediated proliferation and induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes

Rheumatoid arthritis fibroblast-like synoviocytes (RAFLS) proliferate abnormally and resist apoptosis. Geldanamycin (GA) and other HSP90 inhibitors have emerged as promising therapeutic agents that inhibited cancer cell growth. In this study, we explored the effects of HSP90 inhibitor, GA, on tumor necrosis factor (TNF)-α-induced proliferation and apoptosis of RAFLS, and the underlying mechanism. Human RAFLS was isolated from the knee joints of patients with RA and subjected to TNF-α treatment in combination of various concentration of GA. We found that GA dose-dependently inhibited TNF-α-induced RAFLS proliferation as measured, but promoted RAFLS apoptosis. Further mechanistic study identified that GA dose-dependently attenuated TNF-α-mediated activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) pathways, both of which are involved in TNF-α-mediated RAFLS proliferation. Moreover, GA-induced apoptosis and mitochondrial damage of RAFLS, as evidenced by increased Bax/Bcl-2 ratio and mitochondrial cytochrome c release, and enhanced cleavages of caspase-3, caspase-9, and poly-(ADP-ribose) polymerase. Collectively, our results revealed that chemical inhibition of HSP90 by GA suppressed TNF-α-induced proliferation of RAFLSs through the MAPK and NF-κB signaling pathways and induces RAFLS apoptosis via mitochondria-dependent pathway. These findings demonstrated for the first time that HSP90 inhibition in RAFLS could be therapeutic beneficial for RA.     

Three-dimensional constructs induce high cellular activity: Structural stability and the specific production of proteins and cytokines

Naofen has recently been identified from the rat brain/spinal cord cDNA library as a substance reactive against an anti-shigatoxin (Stx)-2 antibody. Naofen mRNA is composed of 4620 nucleotides and encodes 1170 amino acids. Naofen contains four WD-repeat domains in its N-terminus and is ubiquitously distributed in many tissues of the rat. Tumor necrosis factor (TNF)-α enhanced the expression of naofen mRNA in HEK293 cells in a dose-dependent manner. Furthermore, naofen siRNA, which predominantly knocked down the expression of naofen mRNA, significantly reduced both TNF-α-induced caspase-3 activation and apoptosis in HEK293 cells. Overexpression of naofen in HEK293 cells (FLAG-NF) spontaneously induced caspase -3 activation and apoptosis, and showed extremely high susceptibility to TNF-α-induced apoptosis. These results indicated that naofen may function as a novel modulator activating caspase-3, and promoting TNF-α-stimulated apoptosis.     

β-1,4-Galactosyltransferase I involved in Schwann cells proliferation and apoptosis induced by tumor necrosis factor-alpha via the activation of MAP kinases signal pathways

β-1,4-galactosyltransferase-I (β-1,4-GalT-I) plays a critical role in the initiation and maintenance of peripheral nervous system inflammatory reaction. However, the exact function of β-1,4-GalT-I in the regulation of SCs proliferation and apoptosis remains unclear. In this study, we found that low concentration of tumor necrosis factor-alpha (TNF-α) induced SCs proliferation, while high concentration of TNF-α induced SCs apoptosis. Meanwhile, the expressions of β-1,4-GalT-I, TNFR1, and TNFR2 were changed following. When β-1,4-GalT I overexpression, low concentration of TNF-α-induced SCs proliferation was partially repressed. Concurrently, the activity of ERK1/2 was decreased. While knocking down β-1,4-GalT I expression, high concentration of TNF-α-induced SCs apoptosis was partially rescued. Consistent with this, the activity of P38 and JNK were decreased. We also found anti-TNFR2 antibody suppressed low concentration of TNF-α-induced SCs proliferation, while anti-TNFR1 antibody inhibited high concentration of TNF-α-induced SCs apoptosis. Thus, present data show that β-1,4-GalT I may play an important role in SCs proliferation and apoptosis induced by TNF-α via different signal pathways and TNFR.     

Pertussis Toxin, an Inhibitor of G(αi) PCR, Inhibits Bile Acid- and Cytokine-Induced Apoptosis in Primary Rat Hepatocytes

Excessive hepatocyte apoptosis is a common event in acute and chronic liver diseases leading to loss of functional liver tissue. Approaches to prevent apoptosis have therefore high potential for the treatment of liver disease. G-protein coupled receptors (GPCR) play crucial roles in cell fate (proliferation, cell death) and act through heterotrimeric G-proteins. G(αi)PCRs have been shown to regulate lipoapoptosis in hepatocytes, but their role in inflammation- or bile acid-induced apoptosis is unknown. Here, we analyzed the effect of inhibiting G(αi)PCR function, using pertussis toxin (PT), on bile acid- and cytokine-induced apoptosis in hepatocytes. Primary rat hepatocytes, HepG2-rNtcp cells (human hepatocellular carcinoma cells) or H-4-II-E cells (rat hepatoma cells) were exposed to glycochenodeoxycholic acid (GCDCA) or tumor necrosis factor-α (TNFα)/actinomycin D (ActD). PT (50-200 nmol/L) was added 30 minutes prior to the apoptotic stimulus. Apoptosis (caspase-3 activity, acridine orange staining) and necrosis (sytox green staining) were assessed. PT significantly reduced GCDCA- and TNFα/ActD-induced apoptosis in rat hepatocytes (-60%, p<0.05) in a dose-dependent manner (with no shift to necrosis), but not in HepG2-rNtcp cells or rat H-4-II-E cells. The protective effect of pertussis toxin was independent of the activation of selected cell survival signal transduction pathways, including ERK, p38 MAPK, PI3K and PKC pathways, as specific protein kinase inhibitors did not reverse the protective effects of pertussis toxin in GCDCA-exposed hepatocytes. Conclusion: Pertussis toxin, an inhibitor of G(αi)PCRs, protects hepatocytes, but not hepatocellular carcinoma cells, against bile acid- and cytokine-induced apoptosis and has therapeutic potential as primary hepatoprotective drug, as well as adjuvant in anti-cancer therapy.     

Cryptotanshinone enhances TNF-??-induced apoptosis in chronic myeloid leukemia KBM-5 cells

Cryptotanshinone is a biologically active compound from the root of Salvia miltiorrhiza. In the present study, we investigated the molecular mechanisms by which cryptotanshinone is in synergy with tumor necrosis factor-alpha (TNF-α) for the induction of apoptosis in human chronic myeloid leukemia (CML) KBM-5 cells. The co-treatment of cryptotanshinone with TNF-α reduced the viability of the cells [combination index (CI) < 1]. Concomitantly, the co-treatment of cryptotanshinone and TNF-α elicited apoptosis, manifested by enhanced the number of terminal deoxynucleotide transferase-mediated dUTP-nick-end labeling (TUNEL)-positive cells, the sub-G1 cell populations, and the activation of caspase-8 and -3, in comparison with the treatment with either drug alone. The treatment with cryptotanshinone further suppressed TNF-α-mediated expression of c-FLIP(L), Bcl-x(L), but the increased level of tBid (a caspase-8 substrate). Furthermore, cryptotanshinone activated p38 but not NF-κB in TNF-α-treated KBM-5 cells. The addition of a specific p38 MAPK inhibitor SB203580 significantly attenuated cryptotanshinone/TNF-α-induced apoptosis. The combination treatment of cryptotanshinone and TNF-α also stimulated the reactive oxygen species (ROS) generation. N-acetyl-L-cysteine (NAC, a ROS scavenger) was not only able to block cryptotanshinone/TNF-α-induced ROS production but also the activation of caspase-8 and p38 MAPK. Overall, our findings suggest that cryptotanshinone can sensitize TNF-α-induced apoptosis in human myeloid leukemia KBM-5 cells, which appears through ROS-dependent activation of caspase-8 and p38.     

Retinoic acid receptor-mediated signaling protects cardiomyocytes from hyperglycemia induced apoptosis: role of the renin-angiotensin system

Diabetes mellitus (DM) is a primary risk factor for cardiovascular diseases and heart failure. Activation of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) has an anti-diabetic effect; but, a role in diabetic cardiomyopathy remains unclear. Using neonatal and adult cardiomyocytes, we determined the role of RAR and RXR in hyperglycemia-induced apoptosis and expression of renin-angiotensin system (RAS) components. Decreased nuclear expression of RARα and RXRα, activation of apoptotic signaling and cell apoptosis was observed in high glucose (HG) treated neonatal and adult cardiomyocytes and diabetic hearts in Zucker diabetic fatty (ZDF) rats. HG-induced apoptosis and reactive oxygen species (ROS) generation was prevented by both RAR and RXR agonists. Silencing expression of RARα and RXRα, by small interference RNA, promoted apoptosis under normal conditions and significantly enhanced HG-induced apoptosis, indicating that RARα and RXRα are required in regulating cell apoptotic signaling. Blocking angiotensin type 1 receptor (AT(1) R); but, not AT(2) R, attenuated HG-induced apoptosis and ROS generation. Moreover, HG induced gene expression of angiotensinogen, renin, AT(1) R, and angiotensin II (Ang II) synthesis were inhibited by RARα agonists and promoted by silencing RARα. Activation of RXRα, downregulated the expression of AT(1) R; and RXRα silencing accelerated HG induced expression of angiotensinogen and Ang II synthesis, whereas there was no significant effect on renin gene expression. These results indicate that reduction in the expression of RARα and RXRα has an important role in hyperglycemia mediated apoptosis and expression of RAS components. Activation of RAR/RXR signaling protects cardiomyocytes from hyperglycemia, by reducing oxidative stress and inhibition of the RAS.     

Mitochondria-targeted antioxidants prevent TNFα-induced endothelial cell damage

Increased serum level of tumor necrosis factor α (TNFα) causes endothelial dysfunction and leads to serious vascular pathologies. TNFα signaling is known to involve reactive oxygen species (ROS). Using mitochondria-targeted antioxidant SkQR1, we studied the role of mitochondrial ROS in TNFα-induced apoptosis of human endothelial cell line EAhy926. We found that 0.2 nM SkQR1 prevents TNFα-induced apoptosis. SkQR1 has no influence on TNFα-dependent proteolytic activation of caspase-8 and Bid, but it inhibits cytochrome c release from mitochondria and cleavage of caspase-3 and its substrate PARP. SkQ analogs lacking the antioxidant moieties do not prevent TNFα-induced apoptosis. The antiapoptotic action of SkQR1 may be related to other observations made in these experiments, namely SkQR1-induced increase in Bcl-2 and corresponding decrease in Bax as well as p53. These results indicate that mitochondrial ROS production is involved in TNFα-initiated endothelial cell death, and they suggest the potential of mitochondria-targeted antioxidants as vasoprotectors.     

Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.     

Tumor Necrosis Factor-α-Induced Apoptosis in a Human Keratinocyte Cell Line (HaCaT) Is Counteracted by Transforming Growth Factor-α

The integrity of the human epidermis is guaranteed by a regulated balance of proliferation, differentiation, and physiologic cell death of its main cellular constituent, the epidermal keratinocyte. Physiologic cell death is known as apoptosis and has been recognized as an active regulatory mechanism, complementary to, but functionally opposite of, proliferation. The regulators of the delicate balance between cell death and proliferation are only partially understood in human keratinocytes. Transforming growth factor-α (TGF-α) has been identified as a positive regulator of proliferation and growth, while tumor necrosis factor-α (TNF-α) induces apoptosis. Both mediators are thought to influence epidermal keratinocytes under various physiological and pathophysiological conditions. In the current study we have begun to investigate potential regulatory interactions between these two mediators in the human keratinocyte cell line HaCaT. We have found that, when the HaCaT cells were sensitized by the translation inhibitor cycloheximide, TNF-α induced apoptosis, as evidenced by nuclear disintegration, DNA fragmentation (“DNA laddering”), and the appearance of soluble DNA/histone complexes. Moreover, we found that the induction of apoptosis was reduced by preincubation of the cells with TGF-α. The protective effect of TGF-α was abrogated by translation inhibition, indicating that it depended onde novoprotein synthesis. Moreover, the protective effect was not accompanied by a reduced surface expression of TNF receptor molecules. We postulate that TNF-α-induced apoptosis in HaCaT cells is counteracted by constitutively produced suppressors of apoptosis, the synthesis of which can be downregulated by inhibition of translation and upregulated by the cytokine TGF-α.     

Inhibition by Bacterial Lipopolysaccharide of Spontaneous and TNF-α-Induced Human Neutrophil Apoptosis In Vitro

In the previous paper (Takeda et al, Int. Immunol., 5, 691-694, 1993), we demonstrated that tumor necrosis factor-α (TNF-α) promptly accelerates apoptosis of human neutrophils in vitro. In order to determine the role of neutrophil apoptosis in defending against bacterial infection, we studied the effect of bacterial lipopolysaccharide (LPS) on this process. LPS inhibited spontaneous and TNF-α-induced human neutrophil apoptosis in vitro, as determined by 1) light and electron microscopy, 2) flow cytometry, and 3) agarose gel electrophoresis of DNA. Low concentrations of cycloheximide, a protein synthesis inhibitor, which alone did not affect neutrophil apoptosis, were able to reduce spontaneous apoptosis inhibition by LPS, suggesting the involvement of newly synthesized protein in this phenomenon.     

Gsdma3 is a new factor needed for TNF-α-mediated apoptosis signal pathway in mouse skin keratinocytes

Gsdma3, a newly found gene, is expressed restrictedly in mouse skin keratinocytes and gastrointestinal tract. But until now, there is little information on the regulation and the function of Gsdma3 in skin keratinocytes. In our previous study, we found that Gsdma3 mutation resulted in a decrease in catagen-associated apoptosis of hair follicle keratinocytes. Apoptosis of skin keratinocytes is strictly regulated by a series of signal pathways, among of which, tumor necrosis factor (TNF)-α-induced signal pathway has been extensively studied. To further investigate the role and the pathway of Gsdma3 involved in skin keratinocyte apoptosis, using immunofluorescence, RT-PCR, western blot and TUNEL analysis, we showed here that accompanying TNF-α-induced apoptosis and Caspase-3 expression in mouse skin keratinocytes in vivo and in vitro, Gsdma3 expression was significantly upregulated. After Gsdma3 gene mutation, TNF-α-induced apoptosis and Caspase-3 expression in skin keratinocytes were reduced. The injection of Gsdma3 expression plasmid could directly enhance the apoptosis and Caspase-3 expression in skin keratinocytes. These results, taken together, indicated that in mouse skin keratinocytes, Gsdma3 expression could be regulated by TNF-α. Gsdma3 was not only involved in but also necessary for the TNF-α-induced apoptosis pathway by directly enhancing the Caspase3 expression as well as the apoptosis induction.