Angiopoietin-like 4 Enhances the Proliferation and Migration of Tendon Fibroblasts.

PURPOSE: Angiopoietin-like 4 (ANGPTL4) is known to play a variety of roles in the response to exercise, and more recently has been shown to enhance the healing of tendon, a fibrous load-bearing tissue required for efficient movement. The objective of the current study was to further explore the mechanisms of ANGPTL4's effect on tendon cells using a gene array approach. METHODS: Human tendon fibroblasts were treated with recANGPTL4 and their global transcriptome response analyzed after 4 and 24 h. We also conducted functional studies using tendon fibroblasts derived from human subjects, cultured in the presence or absence of applied cyclic stretch and/or siRNA for ANGPTL4, and as confirmation we also used tendon cells from wild type (ANGPTL4 +/+) or knockout (ANGPTL4-/-) mice. RESULTS: The leading functions of ANGPTL4 predicted by the resulting pathway analysis were cell movement and proliferation. The experiments demonstrated that ANGPTL4 significantly enhanced tendon cell proliferation and the cell cycle progression, as well as adhesion and migration. CONCLUSION: Taken together, these findings provide novel molecular insights into the effect of ANGPTL4, a multifunctional protein that regulates the physiological response to exercise, on fundamental tendon cell functions.

The small molecule 2-phenylethynesulfonamide induces covalent modification of p53.

p53 is a tumor suppressor protein which is either lost or inactivated in a large majority of tumors. The small molecule 2-phenylethynesulfonamide (PES) was originally identified as the inhibitor of p53 effects on the mitochondrial death pathway. In this report we demonstrate that p53 protein from PES-treated cells was detected in reduced mobility bands between molecular weights 95-220 kDa. Resolution of p53 aggregates on urea gel was unable to reduce the high molecular weight p53 aggregates, which were shown to be primarily located in the nucleus. Therefore, our data suggest that PES exerts its effects through covalent cross-linking and nuclear retention of p53.

Influence of repetitive mechanical loading on MMP2 activity in tendon fibroblasts.

Matrix metalloproteinase2 has been implicated in tendon pathology caused by repetitive movements. However, its activity in the early stages of the tendon's response to overuse, and its presence in the circulation as a possible indicator of tendon degradation, remain unknown. Human tendon cells were repetitively stretched for 5 days, and the rabbit Achilles tendon complex underwent repetitive motion 3× per week for 2 weeks. Quantitative polymer chain reaction analysis was performed to detect matrix metalloproteinase2/14 and tissue inhibitor of matrix metalloproteinase2 messenger ribonucleic acid of cells and rabbit tissue, and matrix metalloproteinase2 protein levels were determined with an enzyme linked immunoassay. Matrix metalloproteinase2 activity was examined using zymography of the conditioned media, tendon and serum. Immunohistochemistry was used to localize matrix metalloproteinase2 in tendon tissue, and the density of fibrillar collagen in tendons was examined using second harmonic generation microscopy. Tendon cells stretched with high strain or high frequency demonstrated increased matrix metalloproteinase2 messenger ribonucleic acid and protein levels. Matrix metalloproteinase2 activity was increased in the rabbit Achilles tendon tissue at weeks 1 and 2; however, serum activity was only increased at week 1. After 2 weeks of exercise, the collagen density was lower in specific regions of the exercised rabbit Achilles tendon complex. Matrix metalloproteinase2 expression in exercised rabbit Achilles tendons was detected surrounding tendon fibroblasts. Repetitive mechanical stimulation of tendon cells results in a small increase in matrix metalloproteinase2 levels, but it appears unlikely that serum matrix metalloproteinase2 will be a useful indicator of tendon overuse injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1991-2000, 2016.

Etoposide induces cell death via mitochondrial-dependent actions of p53.

BACKGROUND: Etoposide has been used clinically in cancer treatment, as well as in numerous research studies, for many years. However, there is incomplete information about its exact mechanism of action in induction of cell death. METHODS: Etoposide was compared at various concentrations to characterize the mechanisms by which it induces cell death. We investigated its effects on mouse embryonic fibroblasts (MEFs) and focused on both transcriptional and non-transcriptional responses of p53. RESULTS: Here we demonstrate that treatment of MEFs with higher concentrations of etoposide induce apoptosis and activate the transcription-dependent functions of p53. Interestingly, lower concentrations of etoposide also induced apoptosis, but without any evidence of p53-dependent transcription up-regulation. Treatment of MEFs with an inhibitor of p53, Pifithrin-α, blocked p53-dependent transcription but failed to rescue the cells from etoposide-induced apoptosis. Treatment with PES, which inhibits the mitochondrial arm of the p53 pathway inhibited etoposide-induced cell death at all concentrations tested. CONCLUSIONS: We have demonstrated that transcriptional functions of p53 are dispensable for etoposide-induced cell death. The more recently characterized effects of p53 at the mitochondria, likely involving its interactions with BCL-2 family members, are thus more important for etoposide's actions.

Prevention of cytokine withdrawal-induced apoptosis by Mcl-1 requires interaction between Mcl-1 and Bim.

Growth factor withdrawal from hemopoietic cells results in activation of the mitochondrial pathway of apoptosis. Members of the Bcl-2 family regulate this pathway, with anti-apoptotic members counteracting the effects of pro-apoptotic members. We investigated the effect on Mcl-1 function of mutation at a conserved threonine 163 residue (T163) in its proline, glutamate, serine, and threonine rich (PEST) region. Under normal growth conditions, Mcl-1 half-life increased with alteration of T163 to glutamic acid, but decreased with mutation to alanine. However, both T163 mutants exhibited greater pro-survival effects compared with the wild type, which can be explained by an increased stability of the T163A mutant in cytokine-starved conditions. Both the mutant forms exhibited prolonged binding to pro-apoptotic Bim in cytokine-deprived cells. The extent to which Mcl-1 mutants were able to exert their anti-apoptotic effects correlated with their ability to associate with Bim. We further observed that primary bone marrow derived macrophages survived following cytokine withdrawal as long as Bim and Mcl-1 remained associated. In our study, we were unable to detect a role for GSK-3-mediated regulation of Mcl-1 expression. Based on these results we propose that upon cytokine withdrawal, survival of hemopoietic cells depends on association between Mcl-1 and Bim. Furthermore, alteration of T163 of Mcl-1 may change the protein such that its association with Bim is affected, resulting in prolonged association and increased survival.

MCL-1 localizes to sites of DNA damage and regulates DNA damage response.

MCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1(-/-) MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G(1)/S checkpoint response, was not significantly different. In addition, appearance of gamma-H2AX was delayed in the Mcl-1(-/-) MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with gamma-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide , followed by periods of recovery, which mimics the clinical situation of etoposide use, resulted in greater accumulation of chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.

An essential role for MCL-1 in ATR-mediated CHK1 phosphorylation.

Here we report a novel role for myeloid cell leukemia 1 (Mcl-1), a Bcl-2 family member, in regulating phosphorylation and activation of DNA damage checkpoint kinase, Chk1. Increased expression of nuclear Mcl-1 and/or a previously reported short nuclear form of Mcl-1, snMcl-1, was observed in response to treatment with low concentrations of etoposide or low doses of UV irradiation. We showed that after etoposide treatment, Mcl-1 could coimmunoprecipitate with the regulatory kinase, Chk1. Chk1 is a known regulator of DNA damage response, and its phosphorylation is associated with activation of the kinase. Transient transfection with Mcl-1 resulted in an increase in the expression of phospho-Ser345 Chk1, in the absence of any evidence of DNA damage, and accumulation of cells in G2. Importantly, knockdown of Mcl-1 expression abolished Chk1 phosphorylation in response to DNA damage. Mcl-1 could induce Chk1 phosphorylation in ATM-negative (ataxia telangectasia mutated) cells, but this response was lost in ATR (AT mutated and Rad3 related)-defective cells. Low levels of UV treatment also caused transient increases in Mcl-1 levels and an ATR-dependent phosphorylation of Chk1. Together, our results strongly support an essential regulatory role for Mcl-1, perhaps acting as an adaptor protein, in controlling the ATR-mediated regulation of Chk1 phosphorylation.

Cytokine-mediated FOXO3a phosphorylation suppresses FasL expression in hemopoietic cell lines: investigations of the role of Fas in apoptosis due to cytokine starvation.

We have investigated phosphatidylinositol 3-kinase (PI3K)-dependent survival signalling pathways using several cytokines in three different hemopoietic cell lines, MC/9, FDC-P1, and TF-1. Cytokines caused PI3K- and PKB-dependent phosphorylation of FOXO3a (previously known as FKHRL1) at three distinct sites. Following cytokine withdrawal or PI3K inhibition, both of which are known to lead to apoptosis, there was a loss of FOXO3a phosphorylation, and a resulting increase in forkhead transcriptional activity, along with increased expression of Fas Ligand (FasL), which could be detected at the cell surface. Concurrently, an increase in cell surface expression of Fas was also detected. Despite the presence of both FasL and Fas, there was no detectable evidence that activation of Fas-mediated apoptotic events was contributing to apoptosis resulting from cytokine starvation or inhibition of PI3K activity. Thus, inhibition of FOXO3a activity is mediated by the PI3K-PKB pathway, but regulation of FasL is not the primary means by which cell survival is regulated in cytokine-dependent hemopoietic cells. We were also able to confirm increased expression of known FOXO3a targets, Bim and p27kip1. Together, these results support the conclusion that mitochondrial-mediated signals play the major role in apoptosis of hemopoietic cells due to loss of cytokine signalling.

A proteolytic fragment of Mcl-1 exhibits nuclear localization and regulates cell growth by interaction with Cdk1.

Mcl-1 (myeloid cell leukaemia-1) is a Bcl-2 family member with short-term pro-survival functions but whose other functions, demonstrated by embryonic lethality of knockout mice, do not involve apoptosis. In the present study, we show a cell-cycle-regulatory role of Mcl-1 involving a shortened form of the Mcl-1 polypeptide, primarily localized to the nucleus, which we call snMcl-1. snMcl-1 interacts with the cell-cycle-regulatory protein Cdk1 (cyclin-dependent kinase 1; also known as cdc2) in the nucleus, and Cdk1 bound to snMcl-1 was found to have a lower kinase activity. The interaction with Cdk1 occurs in the absence of its cyclin partners and is enhanced on treatment of cells with G2/M blocking agents, but not by G1/S blocking. The snMcl-1 polypeptide is present during S and G2 phases and is negligible in G1. Overexpression of human Mcl-1 in a murine myeloid progenitor cell line resulted in a lower rate of proliferation. Furthermore, Mcl-1-overexpressing cells had lower total Cdk1 kinase activity compared with parental cells, in both anti-Cdk1 and anti-cyclin B1 immunoprecipitates. The latter results suggest that binding to snMcl-1 alters the ability of Cdk1 to bind its conventional partner, cyclin B1. Given the important role of Cdk1 in progression through G2 and M phases, it is probable that the inhibition of Cdk1 activity accounts for the inhibitory effect of Mcl-1 on cell growth.