Phenotype In Human Fibroblasts Research Articles

ANTXR1 deficiency promotes fibroblast senescence: implications for GAPO syndrome as a progeroid disorder

ANTXR1 is one of two cell surface receptors mediating the uptake of the anthrax toxin into cells. Despite substantial research on its role in anthrax poisoning and a proposed function as a collagen receptor, ANTXR1’s physiological functions remain largely undefined. Pathogenic variants in ANTXR1 lead to the rare GAPO syndrome, named for its four primary features: Growth retardation, Alopecia, Pseudoanodontia, and Optic atrophy. The disease is also associated with a complex range of other phenotypes impacting the cardiovascular, skeletal, pulmonary and nervous systems. Aberrant accumulation of extracellular matrix components and fibrosis are considered to be crucial components in the pathogenesis of GAPO syndrome, contributing to the shortened life expectancy of affected individuals. Nonetheless, the specific mechanisms connecting ANTXR1 deficiency to the clinical manifestations of GAPO syndrome are largely unexplored. In this study, we present evidence that ANTXR1 deficiency initiates a senescent phenotype in human fibroblasts, correlating with defects in nuclear architecture and actin dynamics. We provide novel insights into ANTXR1's physiological functions and propose GAPO syndrome to be reconsidered as a progeroid disorder highlighting an unexpected role for an integrin-like extracellular matrix receptor in human aging.

Urolithin A protects human dermal fibroblasts from UVA-induced photoaging through NRF2 activation and mitophagy.

Photoaging, caused by exposure to sunlight and especially UVA, has been identified as one of the culprits for age-related skin deterioration. Here, we initially demonstrated that urolithin A (UroA), a metabolite derived from intestine microflora, possessed sufficient photoprotective capacity and attenuated UVA-induced senescent phenotypes in human fibroblasts, such as growth inhibition, senescence-associated β-galactosidase activity, breakdown of extracellular matrix, synthesis of senescence-associated secretory phenotypes and cell cycle arrest. Furthermore, UroA lessened the accumulation of intracellular reactive oxygen species, which promoted the phosphorylation and afterwards nuclear translocation of NRF2, subsequently driving the activation of downstream antioxidative enzymes. In parallel, we proved that UroA restored mitochondrial function by induction of mitophagy, which was regulated by the SIRT3-FOXO3-PINK1-PARKIN network. Taken together, our results showed that UroA protected dermal fibroblast from UVA damage through NRF2/ARE activation and mitophagy process, thus supporting UroA as a potential therapeutic agent for photoaging.

Ribosomal protein RPL22/eL22 regulates the cell cycle by acting as an inhibitor of the CDK4-cyclin D complex

ABSTRACTSenescence is a tumor suppressor program characterized by a stable growth arrest while maintaining cell viability. Senescence-associated ribogenesis defects (SARD) have been shown to regulate senescence through the ability of the ribosomal protein S14 (RPS14 or uS11) to bind and inhibit the cyclin-dependent kinase 4 (CDK4). Here we report another ribosomal protein that binds and inhibits CDK4 in senescent cells: L22 (RPL22 or eL22). Enforcing the expression of RPL22/eL22 is sufficient to induce an RB and p53-dependent cellular senescent phenotype in human fibroblasts. Mechanistically, RPL22/eL22 can interact with and inhibit CDK4-Cyclin D1 to decrease RB phosphorylation both in vitro and in cells. Briefly, we show that ribosome-free RPL22/eL22 causes a cell cycle arrest which could be relevant during situations of nucleolar stress such as cellular senescence or the response to cancer chemotherapy.

Modulation of the Senescence-Associated Inflammatory Phenotype in Human Fibroblasts by Olive Phenols.

Senescent cells display an increase in the secretion of growth factors, inflammatory cytokines and proteolytic enzymes, termed the “senescence-associated-secretory-phenotype” (SASP), playing a major role in many age-related diseases. The phenolic compounds present in extra-virgin olive oil are inhibitors of oxidative damage and have been reported to play a protective role in inflammation-related diseases. Particularly, hydroxytyrosol and oleuropein are the most abundant and more extensively studied. Pre-senescent human lung (MRC5) and neonatal human dermal (NHDF) fibroblasts were used as cellular model to evaluate the effect of chronic (4–6 weeks) treatment with 1 μM hydroxytyrosol (HT) or 10 μM oleuropein aglycone (OLE) on senescence/inflammation markers. Both phenols were effective in reducing β-galactosidase-positive cell number and p16 protein expression. In addition, senescence/inflammation markers such as IL-6 and metalloprotease secretion, and Ciclooxigenase type 2 (COX-2) and α-smooth-actin levels were reduced by phenol treatments. In NHDF, COX-2 expression, Nuclear Factor κ-light-chain-enhancer of activated B cells (NFκB) protein level and nuclear localization were augmented with culture senescence and decreased by OLE and HT treatment. Furthermore, the inflammatory effect of Tumor Necrosis Factor α (TNFα) exposure was almost completely abolished in OLE- and HT-pre-treated NHDF. Thus, the modulation of the senescence-associated inflammatory phenotype might be an important mechanism underlying the beneficial effects of olive oil phenols.

The activation of PPAR\u03b3 by 2,4,6-Octatrienoic acid protects human keratinocytes from UVR-induced damages

Increasing attention is addressed to identify products able to enhance skin photoprotection and to prevent skin carcinogenesis. Several studies have demonstrated that the α-melanocyte stimulating hormone (αMSH), acting on a functional MC1R, provides a photoprotective effect by inducing pigmentation, antioxidants and DNA repair. We discovered a link between αMSH and the nuclear receptor Peroxisome Proliferator-Activated Receptor-γ (PPARγ), suggesting that some of the αMSH protective effects may be dependent on PPARγ transcriptional activity. Moreover, we demonstrated that the activation of PPARγ by the parrodiene 2,4,6-octatrienoic acid (Octa) induces melanogenesis and antioxidant defence in human melanocytes and counteracts senescence-like phenotype in human fibroblasts. In this study, we demonstrate that the activation of PPARγ by Octa exerts a protective effect against UVA- and UVB-induced damage on normal human keratinocytes (NHKs), the major target cells of UV radiation. Octa promotes the antioxidant defence, augments DNA repair and reduces the induction of proteins involved in UV-induced DNA damage response. Our results contribute to deepen the analysis of the αMSH/PPARγ connection and suggest perspectives for the development of new molecules and formulations able to prevent cutaneous UV damage by acting on the different skin cell populations through PPARγ activation.

Efficient direct conversion of human fibroblasts into myogenic lineage induced by co-transduction with MYCL and MYOD1

The skeletal muscle consists of contractile myofibers and plays essential roles for maintenance of body posture, movement, and metabolic regulation. During the development and regeneration of the skeletal muscle tissue, the myoblasts fuse into multinucleated myotubes that subsequently form myofibers. Transplantation of myoblasts may make possible a novel regenerative therapy against defects or dysfunction of the skeletal muscle. It is reported that rodent fibroblasts are converted into myoblast-like cells and fuse to form syncytium after forced expression of exogenous myogenic differentiation 1 (MYOD1) that is a key transcription factor for myoblast differentiation. But human fibroblasts are less efficiently converted into myoblasts and rarely fused by MYOD1 alone. Here we found that transduction of v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog (MYCL) gene in combination with MYOD1 gene induced myoblast-like phenotypes in human fibroblasts more strongly than MYOD1 gene alone. The rate of conversion was approximately 90%. The directly converted myoblasts (dMBs) underwent fusion in an ERK5 pathway-dependent manner. The dMBs also formed myofiber-like structure in vivo after an inoculation into mice at the subcutaneous tissue. The present results strongly suggest that the combination of MYCL plus MYOD1 may promote direct conversion of human fibroblasts into functional myoblasts that could potentially be used for regenerative therapy for muscle diseases and congenital muscle defects.

Abstract POSTER-BIOL-1346: Growth-suppressive effect of WRN RECQ helicase inactivation in breast and ovarian cancer cells

Abstract One important and high-yielding paradigm in the development of novel, targeted anticancer therapies is to identify genes that are not essential to normal cells but that have become critically important to particular cancer cells as they rely on these genes for survival. This concept, sometimes called addiction of cancer cells to specific genes or pathways can be exploited in the clinic because the function of the genes that cancer cells are addicted to can be disrupted using genetic or chemical manipulation, leading to selective killing of cancer cells while normal cells are minimally affected. For example, it was shown that homologous recombination-defective BRCA1-deficient cancer cells rely heavily on other repair pathways such as NHEJ, one of the pathways facilitated by PARP1. This finding has led to clinical trials designed to determine if PARP inhibitor drugs are an effective treatment for patients with BRCA1-deficient breast and ovarian cancer. We have previously discovered, via a high throughput screen, that simultaneous deficiency in BRCA1 and the WRN RECQ helicase resulted in additive growth-suppressive phenotype in human fibroblasts. WRN is a gene mutated in the Werner syndrome of premature aging, and it is a multifunctional exonuclease/helicase involved in maintenance of genomic stability. Congenital loss of WRN also predisposes to sarcomas and melanomas, though interestingly, not to the most common type of age-related cancer – carcinoma. Near-complete inactivation of WRN in human fibroblasts and common cell lines such as sarcoma-derived U2OS and HT1080, is typically non-lethal, though somewhat reduces cellular growth rate and fitness. Following our original observation, we were interested to determine whether loss of WRN is synthetically lethal with loss of BRCA1 in breast and ovarian cancer cell lines, and if loss of WRN restores sensitivity to platinum or impairs growth in platinum resistant clones derived from breast and ovarian cancer cell lines. Our results show that depletion of WRN in BRCA1-negative or positive breast or ovarian cancer cell lines dramatically suppresses their growth. Both in breast and ovarian cancer cells, at least some of this growth suppression could be attributed to the induction of RNAi-triggered cytotoxicity by WRN downregulation. In addition, WRN depletion also hypersensitized the HCC1937 breast cancer cell line to platinum, however this effect of WRN was attenuated in platinum-resistant clones derived in vitro from this line. Strikingly, non-cancerous mammary epithelial line MCF10a showed virtually no growth suppression by WRN depletion, suggesting that breast and ovarian cancer cells may be more reliant on WRN than normal cells of at least some of the relevant epithelial lineages — which is a therapeutically exploitable trait. We are currently following up on this observation using other non-cancerous epithelial cells, such as primary mammary and fallopian tube epithelial cells, as well as primary human keratinocytes. We will present these data as well as the update on reproducing WRN growth-suppressive phenotype using small molecule inhibitors of WRN. Significance: Our results highlight WRN as a potential novel target for development of breast and ovarian cancer therapies. Citation Format: Piri Welcsh, Keffy Kehrli, Paul Lazarchuk, Julia Sidorova. Growth-suppressive effect of WRN RECQ helicase inactivation in breast and ovarian cancer cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1346.

Adipogenic, Chondrogenic and Osteogenic Differentiation of Clonally Derived Human Dermal Fibroblasts

The apparent need of an autologous cell source for tissue engineering applications has led researchers to explore the presence of cells with stem cell plasticity in several human tissues. Dermal fibroblasts (FBs) are easy to harvest, expand in vitro and store, rendering them plausible candidates for cell-based therapies. The aim of the present study was to observe the effects of adipogenic, chondrogenic and osteogenic induction media on the phenotype of human FBs. Human preadipocytes obtained from fat tissue have been proposed as an adult stem cell source with suitable characteristics, and were used as control cells in regard to their differentiation potential. Routine staining, immunohistochemical analysis and alkaline phosphatase assay were employed, in order to study the phenotypic shift. FBs were shown to possess multilineage potential, giving rise to fat-, cartilage- and bone-like cells. To exclude contaminant progenitor cells or cell fusion giving rise to tissue with adipocyte-, chondrocyte- and osteoblast-like cells, single-cell cloning was performed. Single-cell-cloned FBs (sccFBs) displayed a similar differentiation potential as primary-culture FBs. The presence of ‘stem-cell-specific’ surface antigens was analyzed using flow cytometry. The results reveal that sccFBs have several of the markers associated with cells exhibiting stem cell plasticity. The findings presented here are corroborated by the findings of other groups, and suggest the use of human dermal FBs in cell-based therapies for the reconstruction of fat, cartilage and bone.

Iron-citrate can induce cell senescent phenotype in human fibroblasts in vitro

AbstractAfter a number of replications, human diploid fibroblasts (HDFs) in culture lose the ability to divide, become insensitive to further proliferation and enter a state of replicative senescence (RS). Subcytotoxic doses of several stressful agents such as hydrogen peroxide, tertbutylhydroperoxide or ethanol, are able to cause stress-induced premature senescence (SIPS) in HDFs in vitro. Such senescent cells display many features of RS as growth arrest, senescence associated beta-galactosidase (SA beta-gal), cell enlargement and overexpression of several genes (e.g., p21, TGF beta-1,IGFBP3). During ageing, iron accumulates in several tissues in vivo, and also in senescent HDFs in vitro. Due to its redox-active properties, it promotes hydroxyl radical production (Fenton reaction) and eventually leads to cell injury. Free radical reactions are known to cause the accumulation of intracellular damage resulting in ageing. Iron may thus be able to cause SIPS. The main objective of the present study was to investigate whether the exposure of HDFs to a subcytotoxic concentration of iron is able to cause SIPS.

Proteasome inhibitors shorten replicative life span and induce a senescent‐like phenotype of human fibroblasts

The proteasome constitutes the main non-lysosomal cellular protease activity, and plays a crucial role not only in the disposal of unwanted material, but also in the regulation of numerous cellular processes. Previously, we have reported that during the replicative senescence of WI-38 fibroblasts there is a significant impairment in proteasome activity, which probably has important implications in the control of MAPK signaling and cellular proliferation. In this study, we report the potential role of the proteasome in the generation of the senescent phenotype in WI-38 fibroblasts. Our results indicate that inhibition of proteasome activity leads to an impairment in cell proliferation, and a shortening of the life span. The results also indicate that inhibition of the proteasome in young cells induces a premature senescent-like phenotype, as indicated by the increase in senescence-associated beta-galactosidase (SA beta-gal) activity and the abundance of both p21 and collagenase mRNAs, as well as a decreased level of EPC-1 mRNA known markers of cellular senescence, not previously shown to depend on proteasome activity. Together, our results suggest a molecular mechanism for the lack of responsiveness of human cells to growth factors, and point towards a role for the proteasome in the control of the life span of both cells and organisms.

Increased transcription and modified growth state-dependent expression of the plasminogen activator inhibitor type-1 gene characterize the senescent phenotype in human diploid fibroblasts.

The type-1 inhibitor of plasminogen activator (PAI-1) is a major physiologic regulator of pericellular proteolytic activity and, as such, influences matrix integrity, cell-to-substrate adhesion, and cellular proliferation. Excessive accumulation of both PAI-1 mRNA and protein correlates with the progressive acquisition of morphological and growth traits characteristic of the senescent phenotype (Mu and Higgins, 1995, J. Cell. Physiol., 165:647-657). Compared to early-passage IMR-90 human diploid fibroblasts, a late-passage senescence-associated 11-fold elevation in steady-state PAI-1 mRNA content reflected a 15-fold increase in constitutive PAI-1 gene transcription. Differential mRNA stability was not a factor in age-associated PAI-1 overexpression in IMR-90 cells. Upon removal of serum, early-passage human fibroblasts enter into a state of growth arrest with marked down-regulation of PAI-1 synthesis. Rapid induction of both the 3.0- and 2.2-kb PAI-1 mRNA species was evident upon serum-induced "activation" of quiescent early-passage fibroblasts; induced PAI-1 transcripts were maximal at 2 hr post-serum stimulation and declined in late G1 prior to entry into S phase. In contrast, late-passage (p32) fibroblasts maintained a significant level of PAI-1 expression under serum-free culture conditions. Although the PAI-1 gene was further responsive to serum in senescent cells, transcript abundance remained elevated and actually increased over the 12 to 16 hr post-serum addition period (a time when early-passage fibroblasts down-regulate PAI-1 mRNA content). Development of the senescent phenotype in human fibroblasts is associated, therefore, with significant changes in PAI-1 gene regulation. Such reprogramming involves predominantly transcriptional events and results in a marked increase in steady-state PAI-1 transcript abundance involving both the 3.0- and 2.2-kb mRNA species.

Reversion of the invasive phenotype of human fibroblasts by antimessenger oligonucleotide inhibiting the urokinase receptor gene expression

Reversion of the invasive phenotype of human fibroblasts by antimessenger oligonucleotide inhibiting the urokinase receptor gene expression

Concanavalin A produces a matrix-degradative phenotype in human fibroblasts. Induction and endogenous activation of collagenase, 72-kDa gelatinase, and Pump-1 is accompanied by the suppression of the tissue inhibitor of matrix metalloproteinases.

The lectin concanavalin A (ConA) causes fibroblasts to acquire an arborized morphology and to express elevated levels of collagenase. The temporal and mechanistic aspects of ConA regulation of matrix metalloproteinases (MMPs) and the tissue inhibitor of matrix metalloproteinases (TIMP) were characterized in early passage human fibroblasts. Collagenase (MMP-1), measured by functional assays in the absence of TIMP and also as immunoprecipitated [35S]methionine-labeled protein, was increased 10-20-fold following ConA (20 micrograms/ml, 2 x 10(-7) M) treatment for 24-72 h, with active collagenase comprising approximately 20% of the total collagenase activity. By comparison, MMP-2 (72-kDa gelatinase; molecular mass, 72 kDa, +dithiothreitol; 66 kDa, -dithiothreitol), analyzed by enzymography and following affinity purification, was increased less than 2-fold by ConA and was present entirely as an activated, 61-kDa (+dithiothreitol; 59 kDa, -dithiothreitol) form. Northern hybridization analyses revealed that ConA elevated the steady-state mRNA levels for MMPs; collagenase mRNA increased approximately 16-fold, MMP-2 increased 2-fold, and Pump-1, a recently described MMP gene, was induced. Concomitantly, a 10-fold reduction in TIMP protein and mRNA levels by ConA occurred. In comparison, 12-O-tetradecanoylphorbol-13-acetate (50 ng/ml, 8 x 10(-8) M), which also stimulates collagenase expression strongly (greater than 30-fold), elevated TIMP protein and mRNA levels (2- and 3-fold, respectively) and did not affect MMP-2 expression. The changes in MMP and TIMP mRNA levels induced by ConA were blocked by the protein synthesis inhibitor cycloheximide, and the half-lives of collagenase and MMP-2 mRNAs (53 and 46 h, respectively) were unaffected, indicating that ConA exerts its effects transcriptionally, through pathways requiring de novo protein synthesis. Increased transcription of the mmp genes was confirmed by nuclear run-on analyses; mmp-1 transcription was increased by greater than 25-fold, mmp-2 by approximately 3-fold, and Pump-1 by approximately 7-fold. In contrast, Timp gene transcription was reduced by approximately 80%, revealing reciprocal regulation of MMPs and TIMP during the induction of a resorptive cell phenotype. Decreased amounts of collagen and fibronectin, but not of SPARC (secreted protein, acidic and rich in cysteine) in the conditioned medium was the result of MMP activity since steady-state mRNA levels and transcription of the respective matrix protein genes were unaffected by ConA.