The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

Retrospective commentary

The nuclear envelope is composed by the outer nuclear membrane, which faces the cytoplasm and is continuous with the rough endoplasmic reticulum, and the inner nuclear membrane (INM), which faces the nucleoplasm and is underlain by the nuclear lamina, a meshwork structure composed of intermediate filament lamins. The nuclear lamina provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. Vertebrates have three genes for lamins: LMNA (coding for Lamin A and Lamin C generated by alternative splicing), LMB1, coding for Lamin B1 and LMB2, coding for Lamin B2. A layer of heterochromatin is beneath the nuclear lamina, attached by INM integral proteins which are often organized in multimeric complexes that interact with both lamina and chromatin [1]. Most eukaryotic nuclei have euchromatin located predominantly in the internal nucleus, whereas heterochromatin is surrounding the inner side of the nuclear envelope and the nucleolus. This nuclear architecture is maintained by peripheral heterochromatin sequences binding to the nuclear envelope at lamina-associated heterochromatin domains (coined LAD) [2]. LADs are generally gene poor and those genes in the nuclear envelope associated chromatin are typically repressed.

Role of Lamin B receptor on chromatin structure

Among the INM integral proteins, Lamin B receptor (LBR) is the best studied. It preferentially binds to Lamin B1 through a RS domain and a TUDOR domain both located at the N-terminal end. Lamin B1 is involved in the processes of DNA replication, cell cycle progression, and gene silencing through binding to LADs. LBR selectively interacts with heterochromatin, also with the TUDOR domain, inducing chromatin compaction and repressing transcription. Chromatin compaction is mediated by binding to chromatin regions marked by specific histone modifications, such as dimethylated Lys 20 histone H4 (H4K20me2). Although compaction per se leads to transcriptional repression, LBR also recruits transcriptional repressors such as HP1 (reviewed in [3]). LBR is a chimeric protein, having also a sterol reductase activity located at the C-terminal transmembrane domains; with this activity LBR contributes to cholesterol synthesis, as it catalyzes the reduction in the C14 unsaturated bond of lanosterol, a step of the metabolic pathway of cholesterol synthesis [1] (Figure 1). It has been proposed that cholesterol participates in the assembly of nuclear lipid microdomains that may act as platforms for oligomerizing LBR and for chromatin anchoring, or even directly binding to nucleosomes, thus influencing chromatin condensation and regulating gen expression. LBR sterol reductase activity may contribute to INM lipid rafts formation by producing a cholesterol precursor fostering cholesterol or cholesterol-derived products synthesis (reviewed in [3]). Therefore, having LBR both functions within the same protein, makes it able to contribute to nuclear architecture by, on one hand providing necessary cholesterol precursors to assemble nuclear lipid rafts, which recruits LBR, and, on the other hand tethering heterochromatin to the INM.

Integrative cartoon showing that LBR holds together the nuclear envelop and compressed chromatin in undifferentiated and cancerous cells but is depleted upon DNA damage leading to chromatin decompression and cellular senescence.

Figure 1.
Integrative cartoon showing that LBR holds together the nuclear envelop and compressed chromatin in undifferentiated and cancerous cells but is depleted upon DNA damage leading to chromatin decompression and cellular senescence.

LBR interacts with both Lamin B1 and heterochromatin with the TUDOR domain located at the N-terminus end in the nucleoplasm. LBR also contains a RS domain that interacts with chromatin, and a globular domain that recruits transcriptional repressors like HP1. LBR is a chimeric protein that contributes to cholesterol synthesis through its sterol reductase activity located in the transmembrane domains at the C-terminus end in the INM. Local cholesterol synthesis promoted by LBR sterol reductase activity could lead to INM lipid rafts formation, recruiting LBR itself promoting its multimerization. LBR is lost in cancer cells γ-irradiated, which leads to Lamin B depletion, chromatin decompression and induction of cellular senescence; chromatin changes caused by absence of LBR induce SASP genes expression. At the same time, lack of LBR leads to reduced cholesterol synthesis. LBR loss could also occur during physiological aging contributing to cellular senescence establishment. LBR mutations lead to diseases displaying anomalies of blood cells, lethal developmental defects and progeria-like symptoms. ER, endoplasmic reticulum; ONM, outer nuclear membrane; INM, inner nuclear membrane.

Figure 1.
Integrative cartoon showing that LBR holds together the nuclear envelop and compressed chromatin in undifferentiated and cancerous cells but is depleted upon DNA damage leading to chromatin decompression and cellular senescence.

LBR interacts with both Lamin B1 and heterochromatin with the TUDOR domain located at the N-terminus end in the nucleoplasm. LBR also contains a RS domain that interacts with chromatin, and a globular domain that recruits transcriptional repressors like HP1. LBR is a chimeric protein that contributes to cholesterol synthesis through its sterol reductase activity located in the transmembrane domains at the C-terminus end in the INM. Local cholesterol synthesis promoted by LBR sterol reductase activity could lead to INM lipid rafts formation, recruiting LBR itself promoting its multimerization. LBR is lost in cancer cells γ-irradiated, which leads to Lamin B depletion, chromatin decompression and induction of cellular senescence; chromatin changes caused by absence of LBR induce SASP genes expression. At the same time, lack of LBR leads to reduced cholesterol synthesis. LBR loss could also occur during physiological aging contributing to cellular senescence establishment. LBR mutations lead to diseases displaying anomalies of blood cells, lethal developmental defects and progeria-like symptoms. ER, endoplasmic reticulum; ONM, outer nuclear membrane; INM, inner nuclear membrane.

Interestingly, LBR and Lamin A/C tethers are sequentially used during development regulating heterochromatin positioning, gene expression, and cellular differentiation. In early embryonic development and undifferentiated cells, LBR tethers chromatin repressing expression of differentiation genes, but upon differentiation LBR is replaced by Lamin A/C binding proteins (LAP2/Emerin/MAN1), leading to expression of differentiation genes and repression of cell cycle genes [4]. Accordingly, overexpression of LBR deregulates the differentiation of olfactory neurons [5]. LBR also tethers X chromosome during female mammals development, by its association with Xist long noncoding RNA, changing chromosome X overall structure and allowing its interaction with transcriptional repressors to silence gene expression [6,7].

Role of LBR in cellular senescence

As in embryonic cells, in cancerous cells LBR tethers the chromatin [8]. Interestingly, Emilie Lukasova et al. showed that when DNA is damaged by ɣ-radiation in mammary carcinoma MCF7 and osteosarcoma U2OS cells, LBR is lost and the chromatin structure is altered, promoting cellular senescence [9]. Cellular senescence is a cellular state characterized by terminal cell cycle arrest, accompanied by a lack of response to mitotic and apoptotic stimuli; senescent cells display changes in nuclear morphology and accumulate altered lysosomes leading to increased senescence-associated β-galactosidase activity (SA-βGal). Most importantly, senescent cells express and secrete various growth factors, cytokines, chemokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that alter surrounding tissue [10]. Many senescent cells develop changes in chromatin structure, forming specific subnuclear heterochromatic compartments, called senescence-associated heterochromatin foci (SAHF), which potentially silence genes that promote cell cycle progression. SAHF are highly organized structures having constitutive heterochromatin marked with repressive H3K9me3 at the core, which is surrounded by a layer of facultative heterochromatin marked with H3K27me3 [11]. Senescence-associated terminal proliferation arrest is also driven by activation of the pRB/p16INK4a and p53/p21CIP1 tumor suppressor pathways and sustained DNA damage signaling [12]. Since some of the SASP factors promote paracrine cellular senescence or reinforce autocrine senescence, this phenotype limits tumor progression. Some secreted factors promote clearance of senescent cells by immune cells and facilitate tissue repair, having senescent cells a transient function with a beneficial role in the organism. Nevertheless, when senescent cells are no removed, as occurs with aging, some SASP factors cause chronic inflammation and tumor progression. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment [9].

Previous to Lukasovás work mentioned above, it was shown that changes in nuclear morphology displayed by senescent cells were accompanied by Lamin B1 down-regulation in human and murine primary cells induced to senesce by replicative exhaustion, DNA damage or oncogene expression, as well as in cells in vivo, specially in liver of irradiated mice [13]. Interestingly, even though Lamin B1 is globally reduced during cellular senescence, remaining Lamin B1 redistributes. While the major reduction in Lamin B1 occurs mostly in H3K9me3-enriched regions, favoring spatial re-localization of perinuclear heterochromatin and hence promoting SAHF formation, Lamin B1 binds to H3K27me3-enriched regions, correlating with gene repression [14]. Senescent cells extrude fragments of chromatin from the nucleus into the cytoplasm lacking lamin, which distinguish them from micronuclei (fragments of chromosomes or few chromosomes surrounded by nuclear envelop), and therefore were coined cytoplasmic chromatin fragments. The extruded chromatin is damaged (identified by the presence of ɣ-H2AX) and contains H3K27me3, but is 53BP1- and H3K9ac-negative and is eliminated in the cytoplasm by an autophagic/lysosomal pathway [15]. Lukasova et al. observed that in two types of cancerous cells (MCF7 and USO2) induced to senesce by ɣ-irradiation, the expression of both Lamin B1 and LBR was reduced, accompanied by nuclear modifications including blebbing, micronuclei formation and cytoplasmic chromatin fragments. Interestingly, it seems that euchromatin and heterochromatin are extruded form the nucleus independently. For example, the majority of blebs and micronuclei found in MCF7 cells contained low-density chromatin, Lamin B1, Lamin A/C, and generally lacked LBR and heterochromatin markers such as H3K9me3 and HP1ɣ. Therefore these micronuclei probably represent euchromatin released from the nucleus through lamina compromised by a decreased level of Lamin B1. Senescent cells also formed another type of chromatin-containing structures perhaps generated by extrusion of heterochromatin, which were not coated by lamins, although sometimes heterochromatin was attached to LBR; these cytoplasmic chromatin fragments were extruded from the nucleus through ruptures in the Lamin A/C. Actually, several other nuclear envelop proteins are also down-regulated during oncogene-induced senescence [16].

To analyze the effect of LBR depletion over chromatin structure, Lukasova et al. compared the behavior of two kinds of chromosomes: gene-poor chromosomes 18 and 4 and rich-gene chromosome 17 and 11. Gene-poor chromosomes are located closer to the nuclear periphery, while gene-rich chromosomes are closer to the nuclear center. They observed that reduction in LBR expression caused gene-poor chromosomes 18 and 4 to detach from nuclear membrane and accommodated in the nucleoplasm displaying distension, and thus confirming LBR presumed function to tether chromatin to the nuclear membrane. This was an important observation that provided an explanation as to why chromatin organization and gene expression change during senescence transition. Another important observation was that Lamin B1 loss observed in cellular senescence is a consequence of LBR diminution, as Lamin B1 loss was also observed by just silencing the expression of LBR, in the absence of cellular senescence induction [9]. While the reduction in Lamin B1 is mediated by both mRNA decay [13] and autophagy degradation of Lamin B1 protein [17], it is not yet know the mechanism of LBR reduction upon senescence induction, nor how the loss of LBR leads to Lamin B1 decay. Interestingly, LBR decrease preceded the appearance of senescent features, such as SA-βGal activity. Although silencing the expression of LBR by shRNA caused a slight increase of SA-βGal positive cells compared with cells expressing LBR, it had a clear reduction in proliferation, one of the initial features of senescence establishment. These observations lead Lukasova et al. to propose that LBR depletion triggers cellular senescence.

The original contribution of Lukasova et al. lead to the further discovery by En et al. that chromatin changes caused by LBR reduction during cellular senescence induce an up-regulated expression of SASP factors such as IL-6, IL-8 and MMP1 [18]. It was previously known that the expression of SASP genes is induced by several transcription factors, such as NF-κB, C/EBP-β and GATA4, as well as by structural chromatin changes mediated for example by cyclic GMP-AMP synthase (cGAS) — simulator of interferon genes (STING) signaling pathway and SAHF. Interestingly, En et al. showed that promoters of IL-6, IL-8 and MMP1 SASP genes associate with LBR in normally growing cells, but dissociated from them in senescent cells. Even though LBR knockdown induces cellular senescence in some cell lines (fetal lung TIG-7 cells) but not in others (epithelial HeLa, mammary carcinoma MCF-7 and osteosarcoma U2OS cells), down-regulation of LBR1 induced expression of SASP genes in both TIG-7 and HeLa cell types, regardless of the senescent phenotype. Interestingly, En and colleges observed that LBR down expression leads to DNA instability, potentially activating both NF-κB and cGAS-STING pathways, hence fostering SASP expression [18]. Since LBR tethers transcriptional repressive chromatin, its down-regulation during senescence onset potentially leads to derepression of genes through disorganization of heterochromatin. Therefore, chromatin structural changes caused by lack of LBR cause SASP expression, independent of additional senescence signaling pathways (Figure 1).

Role of LBR dysfunction on disease and possibly aging

LBR dysfunction has relevance in several diseases and potentially in physiological aging. Mutations in LBR gene can lead to diseases ranging from harmless anomalies of blood cells to lethal developmental defects. Interestingly, the changes in nuclear morphology that Lukasova et al. observed in ɣ-irradiated cancerous cells, were similar to those described in fibroblasts and myoblasts from Emery–Dreifuss muscular dystrophy, a heterogeneous late-onset disease involving skeletal muscle wasting and heart defects [19]. This disease is caused in a minority of cases by mutations in either Emerin or Lamins A/C. On the other hand, mutations in LBR are known to cause Pelger–Huët Anomaly in humans, which is a hematological condition where the majority of neutrophil granulocytes exhibit a bilobed appearance, instead of the normal 2–4 lobes. Homozygous Pelger–Huët Anomaly sometimes exhibit skeletal defects, developmental problems and mental retardation, although most individuals die before birth. While the blood granulocyte nuclear changes are explainable by the role of LBR tethering heterochromatin to the nuclear envelop, the developmental problems imply that other LBR functions might be responsible. The lack of sterol reductase activity of LBR seems to be the cause of the disease mentioned above, and also of the rare autosomal recessive syndrome HEM/Greenberg skeletal dysplasia, which is embryonic lethal. Skin fibroblast from an 18-week old fetus accumulated a sterol precursor intermediate in cholesterol synthesis, not seen in fibroblast from healthy fetus of the same age, and the mother of HEM fetus had the granulocyte nuclear bilobed classical of Pelger–Huët Anomaly, demonstrating she was heterozygous for the LBR mutation (reviewed in [1]).

A similar phenotype to Pelger–Huët Anomaly is observed in mice called ‘ichthyosis', which are deficient of LBR. Interestingly, ichthyosis mice show skin defects, alopecia, and short life-span, similar to progeria syndromes [20]. In addition, ichthyosis mice have elevated accumulation of advanced glycation end products (AGEs), another feature of aging [21]. This finding suggests that a reduction in LBR could contribute to organismal aging, and perhaps facilitates accumulation of senescent cell in aged individuals (i.e. it not only induces senescence in cancer cells). In this context, it is tempting to speculate that LBR fosters senescence transition by two mechanisms: (1) by altering chromatin structure to give rise to changes that limit cell cycle progression and induce SASP genes expression; and (2) by a reduction in cholesterol availability at the nuclear membrane (Figure 1). That a reduction in cholesterol availability caused by LBR loss could also promote senescence, is supported by the finding that adding cholesterol to bone marrow mesenchymal stem cells induced to senesce by H2O2 exposure or replicative senescence, protects them. Cholesterol anti-senescence effect is mediated by modulating autophagy and the ROS/p53/p21Cip1/Waf1 signaling pathway [22].

Concluding remarks

A main feature of aged organisms is the accumulation of senescent cells, which can reduce regenerative capacities and create a pro-inflammatory microenvironment favorable to the onset and progression of several age-associated diseases, cancer among them. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure and compartmentalization together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

Competing Interests

The author declares that there are no competing interests associated with this manuscript.

Acknowledgements

SCO research on cellular senescence, DNA damage and aging was supported by grants CONACyT FC921 and UNAM/PAPIIT IN206518. Author is thankful to Camila del Rio-Castro (https://camidelrica.wixsite.com/neurocosa-que-dibuja) for preparation of the integrative cartoon, and Dr. Beatriz Aguilar for her technical assistance.

Abbreviations

     
  • AGEs

    advanced glycation end products

  •  
  • INM

    inner nuclear membrane

  •  
  • LBR

    Lamin B receptor

  •  
  • SAHF

    senescence-associated heterochromatin foci

  •  
  • SASP

    senescence-associated secretory phenotype

  •  
  • STING

    simulator of interferon genes

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