The Dual Roles of Lamin A/C in Mechanosensation to Compressive Force

doi:10.21203/rs.3.rs-3586140/v1

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The Dual Roles of Lamin A/C in Mechanosensation to Compressive Force

https://doi.org/10.21203/rs.3.rs-3586140/v1

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Cellular mechanosensation is a complex physiological process coupling alterations in the external environment and cellular behaviors. Over the past decade, the role of the nucleus in mechanosensation has gained increasing attention. Our research found that lamin A/C, a component of the nuclear envelope, plays a dual role in the mechanosensation of macrophages in response to compressive force. Our findings indicated that hydrostatic compressive force downregulated lamin A/C protein via the cytoskeleton. Consequently, this lamin A/C deficiency enhanced compressive-force-induced inflammatory cytokines secretion and proliferative impairment. Unexpectedly, lamin A deficiency also inhibits compressive force-induced DNA damage and interferon regulatory factor 4 (IRF4) up-regulation. Our findings suggest that lamin A/C is involved in multiple mechanosensation mechanisms. Mechanistically, lamin A/C deficiency augments nuclear permeability, facilitates the activation of yes-associated protein 1 (YAP1) and promotes force-induced nuclear translocation of YAP1. These mechanisms have been validated to favor mechanosensation. Conversely, we also found that lamin A/C deficiency led to detachment of components of linker of nucleoskeleton and cytoskeleton (LINC) complex, which impeded intracellular mechanotransmission. In summary, lamin A/C can promote some responses of macrophages to mechanical compression but inhibits others. It is involved in two distinct mechanisms: enhancing nuclear permeability to transcription factors and impairing mechanotransmission by disrupting the LINC complex's connection to the nucleus.

Biological sciences/Cell biology/Cell signalling/Stress signalling

Biological sciences/Cell biology/Nuclear transport/Protein transport/Protein translocation

Macrophage

mechanosensation

compressive force

Lamin A

LINC complex

YAP1

Macrophages, essential innate immune cells, are found ubiquitously in all tissues and body compartments [1]. They exhibit remarkable plasticity and heterogeneity due to dynamic regulation by complex gene networks and signaling cascades [2]. The fate of macrophages plays a pivotal role in various physiological processes, disease and pathology evolution, including inflammation [3], liver diseases [4], atherosclerosis [5], Alzheimer’s disease [6], organ fibrosis [7], cancer [8] among others. Until now, most studies have primarily focused on biochemical stimulation applied to macrophages. However, recent research has revealed that macrophages can also respond to various forms of biomechanical stimulation, such as cyclic tensile force [9], orthodontic compression [10], spatial confinement [11] and matrix stiffness [12]. In our previous study [13] we established an in vitro model that mimicked hydrostatic compression during orthodontic treatment and observed its effects on macrophage polarization, although the upstream mechanotransduction pathway remained elusive.

The nuclear lamina, a protein network approximately 15 nanometers thick located beneath the inner surface of the nuclear envelope, consists of four distinct types of intermediate filaments in mammalian cells: lamin A, B1, B2 and C [14–16]. Lamin A and its splice variant lamin C, both encoded by LMNA, have high similarities in their primary sequences, so they have mostly been studied together as lamin A/C [17]. Given that nuclear lamina is highly conserved and widely expressed in nearly all cell types, defects or deficiencies in nuclear lamina lead to severe consequences. Laminopathies, a group of diseases resulting from mutations in the LMNA, are characterized by premature aging, cardiomyopathy, lipodystrophy, muscular dystrophy, microcephaly, epilepsy, mandibuloacral dysplasia type A, axonal peripheral neuropathy, and more [18, 19]. The nuclear lamina has been believed to undertake two cellular functions. Firstly, it is an essential structure providing mechanical support to the nucleus and protection to genome, an abnormally assembled lamina causes nuclear envelope rupture [20]. Secondly, as large parts of the chromatin bind and interact with it, the nuclear lamina can modulate chromatin organization and the accessibility of signaling molecules and transcription factors to target promoters [15]. In addition to these two functions, the nuclear lamina has also been observed to undergo changes in quantity and conformation in response to mechanical loading [21, 22]. This suggests its potential role as an effector in response to external mechanical stimuli.

Lamin A/C, the predominant constituent of the nuclear lamina, is distributed throughout the nucleoplasm [22, 23]. It connects to the cytoskeleton via the linker of nucleoskeleton and cytoskeleton (LINC) complex. This connection allows lamin A/C to respond to external cues, including tissue stiffness. [22, 24]. Additionally, lamin A/C also interfaces with chromatin, participating diverse cell signaling pathways that influence fundamental cellular processes such as proliferation, migration, genome organization, and DNA repair [25]. Hence, we hypothesize that lamin A/C acts as a mediator, linking external mechanical stimuli to nuclear responses, thereby facilitating cellular mechanotransduction.

1.1 Cell Culture and Compressive Force Loading

The murine macrophage cell line RAW264.7 was purchased from the ATCC. Briefly, the macrophages were cultured in DMEM (41965062, Gibco) with 10% Fetal Bovine Serum (FBS) (10270-106, Gibco) and 1% Penicillin/Streptomycin (A3160502, Gibco). The cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. The cells were seeded into 6-well plates (657160, Greiner Bio-one) at the density of 1×10⁶ cells / well 24 h before compressive force loading. The contactless force-loading appliance utilized has been described in our previous publication [13]. Briefly, a floating weight was placed on the medium to subject macrophages to 1 g / cm² hydrostatic pressure. Cells were harvested by scraper to operate further analysis.

1.2 Small interfering RNA oligonucleotides

The siRNAs targeting mouse LMNA (the gene coding lamin A/C) (SI01090803), negative control (1027280) and cell death control (SI04939025) were purchased from QIAGEN (Germany). Transfection mixture was formulated of 1.2 µl siLMNA oligonucleotides, 12 µl HiPerFect@ Transfection Reagent (301705, QIAGEN) and 100 µl serum-free medium. The mixed liquor was incubated at room temperature (RT) for 10 min before being added to 2.3 ml complete medium in the 6-well plate. Meanwhile, macrophages were seeded in the density of 6 × 10⁵ cells/well. After transfection for 24 hours, 1 g/cm² compressive force was loaded if needed.

1.3 Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)

The total RNA was isolated using the ReliaPrep™ RNA Miniprep Systems (z6011, Promega). RNA concentrations were measured at 260 nm using a spectrophotometer (Nanodrop2000, Thermo Scientific). cDNA was synthesized from 1.0 µg of total RNA using the Verso cDNA Synthesis Kit (AB1453B, Thermo Fisher) and performed on CFX96TM System Cycler (Bio-Rad).

The SsoAdvancedTM Universal SYBR@ Green Supermix (1723271, Bio-Rad) was used in each reaction setup. The primers employed were mouse LMNA (QT00143213, Qiagen), inducible nitric oxide synthases (QT01547980, Qiagen), Arginase 1 (QT00134288, Qiagen), IL-10 (QT00106169, Qiagen), SUN1 (QT00139223, Qiagen), SUN2 (QT00138530, Qiagen). β-Actin (QT00095242, Qiagen) was used as housekeeping gene. Results were analyzed using the Bio-Rad CFX Manager 3.1 software.

1.4 Proteins Extraction and Western Blot Analysis

RIPA buffer (89901, Thermo Scientific) supplemented with 3% protease inhibitor (78442, Thermo Scientific) was used for protein extraction. Protein concentrations were measured using PierceTM BCA Protein Assay Kit (23225, Thermo Scientific) on a direct reading Spectrophotometer (DR/2000, HACH). Further, 20 µg protein samples were separated using 10% SDS-PAGE gel by electrophoresis and transferred to a nitrocellulose membrane (1704271, Bio-Rad). The membranes were blocked with 5% non-fat milk (T145.1, ROTH) in 4 ℃ for 24 h and incubated with the primary antibodies for lamin A/C (ab133256, Abcam), Interferon regulatory factor 4 (SAB4501566, Sigma), activated YAP1 (ab205270, Abcam), SUN1 (ab103021, Abcam), SUN2 (ab124916, Abcam), phosphorylated H2A histone family member X (γH2AX)(ab81299, Abcam), β-actin (ab8227, Abcam) and GAPDH (G8795, Sigma) at a concentration of 1:1,000. The secondary antibodies employed were: Polyclonal Goat Anti-Rabbit (P0448, Dako) and Polyclonal Goat Anti-Mouse (P0447, Dako) at a concentration of 1:2000. The band signals were detected with ChemiDoc Imaging System (Bio-Rad) utilizing Clarity Western ECL Substrate (170–5061, Bio-Rad).

1.5 Comet assay

The comet assay was performed following the manual of the comet assay kit (ab238544, Abcam). Briefly, cells were prepared as single cell suspensions and combined with agarose at 37 ℃. The agarose/cells mixture was pipetted onto a glass slide. After the samples were solidified, they were treated with lysis buffer and alkaline solution included in the kit. Electrophoresis was performed via Tris/Borate/EDTA (TBE) electrophoresis (10.8 g Tris Base, 5.5 g Boric Acid, 0.93g EDTA disodium salt and adjust volume to 1L with DI H₂O) at 2 volt/cm for 15 min. Cells were stained with DNA dye provided in the kit for 15 min and viewed by epifluorescence microscopy using a FITC filter.

1.6 Evaluation of Nuclear Permeability

The evaluation method is modified from the protocol reported by Marcela Raices and Maximiliano A. D’Angelo [26]. The protocol is briefly described as follows: the macrophages were seeded together with the siLMNA into the 6-well plate. After 24 h, cells were rinsed with cold PBS for 2min and incubated with buffer A (20 mM HEPES pH 7.5, 110 mM KOAc, 5 mM MgCl2, 0.25 M sucrose, and protease inhibitors.) for 5 min on the ice. After then, macrophages were incubated with buffer B (20 µg/ml 500 or 65–80 KDa TRITC-dextran, 20 mM HEPES pH 7.5, 110 mM KOAc, 5 mM sodium chloride, 2 mM MgCl₂, 0.25 M sucrose, and protease inhibitors) for 10 min. Cells were rinsed for 3 times with cold PBS and mounted by fluorescent Mounting Medium with DAPI (ab104139, Abcam). Finally, the cells were observed under a confocal laser microscope (Carl Zeiss, Germany) with 547 nm (TRITC) or 365 nm (DAPI) Laser to evaluate nuclear permeability. For quantification using the Image J software (National Institutes of Health and University of Wisconsin, United States), the DAPI coloring area was selected as the region of interest (ROI), the ratio of TRITC integrated density in ROI to entire cells was calculated as the index of permeability.

1.7 Immunofluorescence (IF) Staining

Macrophages were fixed with 4% paraformaldehyde (30525-89-4, Sigma- Aldrich) for 15 min at room temperature. Cells were permeabilized using 0.5% Triton X-100 (28313, Thermo-Fisher) for 10 min. Then, cells were blocked in immunofluorescence blocking buffer (12411S, Cell signaling Technology) for 30 min at room temperature and further incubated with primary antibodies lamin A/C (ab133256, Abcam) (dilution 1:100) and activated YAP1 (ab205270, Abcam) at 4°C overnight. The secondary antibodies that were used included goat anti-rabbit IgG H&L Alex Fluor 594® (ab150080, Abcam) or Alexa Fluor® 488(ab150077, Abcam) After washing with PBS, samples were incubated with phallodin-iFlour 488 Reagent (ab176753, Abcam). After rinsing with PBS for 3 times, all samples were mounted using a fluorescent Mounting Medium with DAPI (ab104139, Abcam). Staining was analyzed using a high-resolution fluorescence microscope (Leica Microsystems, Wetzlar, Germany) or confocal laser microscope (Carl Zeiss, Göttingen, Germany) and photographed.

1.8 Statistical Analysis

Statistical analyses was performed using the GraphPad Prism 9.0 software (GraphPad software, La Jolla, CA, USA). All values are expressed as means ± standard deviation (SD) and were analyzed using one-way ANOVA or Student’s t-test to determine the statistically significant differences between groups. Differences were considered statistically significant at a p value of < 0.05. All experiments were repeated successfully at least three times.

2.1 Lamin A/C protein was down-regulated by Compressive Force

After application of the compressive force, we observed a significant decrease in lamin A/C expression at 1 and 3 hours, with reductions of 35.25% at 1 hour and 33.11% at 3 hours. Notably, lamin A/C levels returned to their original state by the 6-hour mark (Fig. 1a&b).

To investigate the role of the cytoskeleton, the macrophages were pre-incubated with 2 µM cytochalasin D (PHZ1063, ThermoFisher), a cytoskeleton-disrupting agent that induces F-actin depolymerization [27, 28], before subjecting them to compressive force. The western blot analysis showed no significant difference among all time points (Fig. 1c&d), suggesting that cytochalasin D effectively eliminated the impact of compressive force on lamin A/C.

Next, we stained the macrophages with an anti-lamin A/C fluorescent antibody and observed them using confocal laser fluorescence. The fluorescence intensity of lamin A/C exhibited a significant decrease at 3 hours (Fig. 1e&f). Additionally, a considerable proportion of macrophage nuclei displayed abnormal morphology, resembling shrunken peanut-like structures. However, this abnormality partially returned to normal at 3 hours (Fig. 1e). Importantly, there was no alteration in gene levels at either 1 or 3 hours. These findings suggest that compressive force rapidly reduces lamin A/C levels, likely acting directly on the protein rather than affecting the gene transcription.

2.2 Lamin A/C Deficiency enhanced Effects of Compressive Force on proliferation and inflammatory gene expression

To achieve a more sustained deficiency in lamin A/C, we transfected macrophages with siRNA. qRT-PCR analysis revealed a significant decrease in LMNA mRNA levels at 24- and 48-hours post-transfection, although the inhibitory effect vanished after 72 hours (Fig. 2a). Consistently, immunofluorescence (IF) staining revealed that the lamin A/C protein levels decreased at 24- and 48-hours post-transfection but returned to baseline levels after 72 hours (Fig. 2b). This effect suggests that siLMNA effectively maintains low protein levels for a duration of 24 to 48 hours following transfection. Hence, we subjected the cells to 1g/cm² compressive force at 24 hours post-transfection and harvested them after 48 hours for further protein, proliferation, and genetic analysis. Results from the Western blot experiments confirmed that siRNA induced significant lamin A/C deficiency, whether with or without compression loading (Fig. 2c & d). On the other hand, we applied 1 g/cm² compressive force on macrophage. In line with our previous study [13], compression prompted gene expressions of Arg1, Il10 and NOS2, suggesting the inflammatory activation of macrophages (Fig. 2f-h). Meanwhile, compression also inhibited cellular proliferation (Fig. 2e). Conversely, independent lamin A/C deficiency did not affect proliferation or inflammatory genes (Fig. 2e), but it significantly enhanced the effect of compression on macrophages (Fig. 2e-h).

2.3 Lamin A/C Deficiency Inhibited Compressive Force-Induced DNA Damage and IRF4 Expression

Considering that both compressive force and siLMNA resulted in nuclear deformation, we assessed their effects on DNA damage. γH2AX, a biomarker for DNA double-strand breaks, was assayed by Western blot. These experiments revealed that compressive force significantly increased the γH2AX levels compared to the control group (p < 0.05). Interestingly, although lamin A/C is known as a nuclear supporter due to its proportional relationship with nuclear stiffness, siLMNA reduced significantly γH2AX protein levels (Fig. 3a&b). We further corroborated these findings with a comet assay. Specifically, cells subjected compressive force had the longest ‘comet tail’ comprised of DNA fragments and siLMNA significantly shortened these tails (Fig. 3c). Therefore, we believed that compressive force elicited DNA damage in macrophage, and this damage was dependent on the presence of lamin A/C. Consistently, our results also demonstrated that compressive force increased interferon regulatory factor 4 (IRF4) levels, a transcription factor associated with the anti-inflammatory activation of macrophages. Notably, the compressive force-induced elevation of IRF4 was significantly inhibited by lamin A/C deficiency (Fig. 3d&e). Interestingly, in contrast to the findings in the previous section, this evidence indicates that lamin A/C reduce some of the effects of compressive force on macrophages. To the best of our knowledge, this is the first report of lamin A/C exhibiting dual roles in cells exposed to compressive force. Hence, we conducted further investigations to elucidate the underlying mechanisms.

2.4 Lamin A/C Deficiency Increased Nuclear Permeability to YAP1

Given that lamin A/C is a primary component of the nuclear lamina, a protein network that supports the inner nuclear envelope and encases the nuclear chromatin, we hypothesized that lamin A/C deficiency might lead to increased nuclear permeability. To evaluate nuclear permeability, we used two types of fluorescent dextrans with different molecular masses. The smaller molecular mass dextran (65–80 kDa) can passively diffuse into the nucleus through the nuclear pore transport complex (NPC), whereas the larger molecular mass (500 kDa) dextran requires NPC deformation or nuclear damage for permeation (Fig. 4a). The lamin A/C deficiency significantly increased permeability of the large molecular dextran (p < 0.001), but had no influence on the smaller molecular dextran (p>0.05) (Fig. 4b-e).

Besides, Yes-associated protein 1 (YAP1) is a well-known mechanically sensitive transcription factor that accumulates in the nucleus under mechanical force [29]. Western-blot results showed that both siLMNA and compressive force increased the level of active YAP1 (Fig. 5a&b). Moreover, immunofluorescence revealed that independent siLMNA or independent compressive force did not affect YAP1 translocation (Fig. 5c&d). However, in the compressive force + siLMNA group, the intranuclear signal was increased 2.5-fold compared to the control group (Fig. 5c&d). These results support the assumption that lamin A/C limits the rate of compressive force-induced YAP1 translocation. Another interesting observation is that cells subjected to compressive force displayed nuclei that appeared to be shrinking, and cells treated with both compressive force and siLMNA exhibited even more pronounced nuclear deformations (Fig. 5d). Therefore, it is reasonable to assume that the nucleus tends to undergo morphological changes under compression, and lamin A/C plays a role as a structural supporter.

2.5 Lamin A/C Deficiency Detached LINC Complex from Nucleus

LINC complex is a vital structure that connects the cytoskeleton to the lamina, playing a critical role in intracellular mechanotransmission [30]. Therefore, we evaluated the impact of lamin A/C deficiency on the LINC complex. Genetic and protein assays demonstrated that neither compressive force nor siLMNA altered the SUN1 level, one of the components of the LINC complex (Fig. 6a-c). Besides, the level of SUN2, another component of the LINC complex, was significantly suppressed by compressive force (p < 0.0001) but remained unaffected by siLMNA (Fig. 6d-f). Moreover, based on immunofluorescent images, SUN1 signals are assembled in the nucleus, but siLMNA and/or compressive force caused SUN1 to diffuse into the cytoplasm (Fig. 6g). Similarly, an intact circle of SUN2 surrounded nucleus was observed in the control group but siLMNA or (and) compressive force resulted in noticeable disruption of the SUN2 circle (Fig. 6g). In summary, only compressive force reduced the protein level of SUN2, but both compressive force and lamin A/C deficiency led to the detachment of SUN1 and SUN2 from the nucleus.

Cellular mechanosensation involves the conversion of mechanical signals into biochemical or electrical signals, which in turn regulate cellular functions, gene expression, and epigenetic programming [31, 32]. Cellular mechanosensation has been established to be associated with extremely wide pathological and physiological processes, including nerve impulses [33], innate immunity [34], tissue morphogenesis [35, 36], wound healing [37], skin hemostasis [38], bone remodeling [39, 40], orthodontic tooth movement [41], cancer progression [42, 43], periodontitis [44], pulmonary fibrosis [45], portal hypertension [46] and atherosclerosis [47]. Therefore, understanding the mechanisms of cellular mechanosensation is of immense significance. To date, the most extensively studied mechanical sensors are various ion channels on the cell membrane. Among them, Piezo1 and Piezo2 stand out as the most famous channels, first reported by Patapoutian and colleagues in 2010 [48]. These channels can be activated by membrane stretching, leading to the influx of cations and initiating downstream mechanotransduction [49]. However, recent research has discovered that the nucleus, located deep within cells, can also directly respond to mechanical force [50, 51]. While our previous research has shown that static compression can upregulate histone acetylation [13] the key link connecting extracellular mechanical force and intranuclear responses remains unidentified.

Our findings have indicated that lamin A/C plays dual roles in cellular mechanosensation. Firstly, we observed that static compressive force leads to a transient downregulation of lamin A/C protein levels without affecting gene expression (Fig. 1a&b). This effect occurs earlier than compressive force-induced inflammatory genes expression [13] and is cytoskeleton-dependent (Fig. 1c&d). Chambliss (2013) [52] and Fu (2023) [53] reported a similar lamin A/C response to various mechanical stimulations. Interestingly, different cell lines may respond to the same mechanical force differently, or even oppositely. Maremonti (2022) [54] observed contrasting responses in two different human epithelial cell lines (MDA-MB-231 and MCF-10A) subjected to the same mechanical stimulation. Anyway, it is convincing that compressive force can act on lamin A/C via the cytoskeleton/lamin A/C axis, a key aspect of mechanotransduction. In addition, the compression-induced lamin A/C deficiency was completely rescued after 6 h according to Western blot analysis (Fig. 1a&b). Numerous evidence has revealed that lamin A/C works dynamically in acclimating external environment and regulating chromatin accessibility in the cell cycle [22, 55, 56]. The lamin A/C level is proportional to nuclear stiffness, and it can be enhanced by some external stimulations, such as a rigid extracellular matrix [22, 57]. Thus, we supposed that compressive force impairs lamin A/C in the early stage, but subsequently initiates a repair mechanism to rescue the lamin A/C level and strengthen the nuclear stiffness to resist the increased external pressure. Lamin A/C deficiency conducted by siRNA was also rescued after 72 h both on the genetic and protein level, providing further support for this theory. Another indirect evidence for our assumption is that lamin A/C is considered as a highly viscous fluid within the "balloon" that hinders nuclear deformation [22]. Immunofluorescence analysis demonstrated significant nuclear deformation occurring within 1 hour of compression loading, gradually reducing despite continued compression. This repair mechanism theory could possibly explain two questions: 1. Why do some cells respond differently to compressive force over time? Our hypothesis is that the repair mechanism following the initial cellular response altered the cellular mechanical effector (lamin A/C). 2. Why do certain cell lines exhibit contrasting responses to the same mechanical stimulation? We propose that the repair mechanism may overcompensate in these cases.

Moreover, the application of compressive force triggers diverse alterations in macrophage behavior. To investigate the role of lamin A/C in mechanosensation, we employed siRNA to silence LMNA genes and reduce the lamin A/C protein level. Surprisingly, the influence of lamin A/C deficiency on these changes is inconsistent (Fig. 7).

Firstly, lamin A/C deficiency significantly intensified force-induced proliferative impairment and the expression of inflammatory genes (Arg1, Il10, and Nos2) (Figs. 2e-h). This finding aligns with the conventional theory that lamin A/C, serving as the nuclear scaffold, counteracts mechanical pressure's impact on the nucleus [58, 59]. This is further supported by the observation that fibroblasts lacking lamin A/C face an increased risk of nuclear rupture under compression [82, 83]. Some of our study's results indirectly support this notion: macrophages exposed to compressive force exhibit significant nuclear shrinkage, and lamin A/C-deficient cells exacerbate this deformation (Fig. 5d & 6g).

However, we also observed that lamin A/C deficiency inhibited force-induced DNA damage and the expression of IRF4 (Figs. 3). This finding contradicts the conventional theory since nuclei lacking lamin A/C protection were expected to withstand higher mechanical forces and a greater risk of damage.

Nevertheless, the diverse effects of lamin A/C deficiency suggest its involvement in two distinct mechanisms, playing contrasting roles in mechanosensation.

Mechanistically, we observed that lamin A/C deficiency significantly increased the nuclear permeability for large dextran (Fig. 4b&c). This probably is caused by forced-induced NPC conformational alteration [60, 61]. Another possible reason is nuclear micro rupture which has also been reported to occur due to mechanical force or cellular senescence [62]. Anyway, we observed that the increased permeability is coincides with enhanced nuclear translocation of YAP1 (Fig. 5c&d). YAP1 is a classic mechanical response protein, numerous studies have uncovered that it mediates the mechanosensation by shuffling into the cellular nucleus [63–68]. Its most well-known regulatory mechanism is the Hippo signaling pathway [69]. In brief, the Hippo signaling pathway is initiated when the large tumor suppressor kinase 1/2 (LATS1/2) complex is phosphorylated, leading to the downstream phosphorylation of YAP1. Phosphorylated YAP1 binds to 14-3-3 proteins and is eventually degraded in the cytoplasm. When the Hippo signaling pathway is 'turned off,' non-phosphorylated YAP1 translocate into the nucleus and triggers gene transcription. Apart from the Hippo pathway, YAP1 translocation can also be modulated by other mechanisms[70–72], and we assume that lamin A can be one of them. Artola et al. (2016) [73] found that fibroblasts growing on soft extracellular matrix, a network of extracellular macromolecules and minerals which provides cells with structural and biochemical support, exhibited greater YAP1 translocation. Meanwhile, Swift et al. (2013) [22] reported that soft extracellular matrix can lower mesenchymal stem cells (MSCs) nuclear lamin A/C level. Their findings have offered indirect evidence supporting association between lamin A and Yap1 translocation. Our data strongly confirmed that compressive force can induce YAP1 nuclear translocation only in macrophage with lamin A/C deficiency, in another word, lamin A/C mechanically inhibits YAP1 translocation. Interestingly, lamin A/C may be not completely Hippo pathway-independent because we observed higher non-phosphorylated YAP1 (activated YAP1) in cells lacking lamin A/C (Fig. 5a&b). However, this lamin A/C deficiency-induced non-phosphorylated YAP1 cannot be accumulated in the nucleus unless compressive force is loaded (Fig. 5a-d). This effect indicates that compressive force provided some initial factor in addition to the non-phosphorylated state, which is required for YAP1 translocation. According to all above evidence, we propose a possible regulated mechanisms of YAP1 translocation by both compressive force and lamin A/C (Fig. 8). However, further exploration of the molecular mechanisms involved is warranted.

Finally, our study has revealed that lamin A/C deficiency leads to detachment of the LINC complex from the nucleus (Fig. 6g). The LINC complex forms a physical connection between cytoskeleton and nucleoskeleton, which transduces the mechanical signals from the environment to the nucleus [74]. The LINC complex consists of an outer nuclear membrane, the Klarsicht/ANC-1/Syne-1 homology (KASH) domain protein and an inner nuclear membrane SUN domain protein. SUN1 and SUN2 constitute the inner nuclear membrane (INM) component of the LINC complex, interacting with lamina and connecting with the KASH domains of nesprins in the perinuclear space (PNS) [75, 76]. Some scholars have reported that the deletion of lamin A/C impairs the nuclear localization of SUN2 [77, 78], which is consistent with our findings. Whereas its effects on SUN1 is controversial. Chiarini et al. (2022) [79] and Mattioli et al. (2011) [80] reported that the presence of lamin A/C is significantly proportional to the nuclear localization of SUN1, but Haque et al. (2010) [78] reported that lamin A/C did not affect SUN1 localization and believed SUN1 was anchored on nucleus by binding to chromatin rather than the lamina. Differing conclusion may be due to different cell lines used. On the other hand, our data shows the lamin A/C deficiency didn’t affect SUN1 or SUN2 expression (Fig. 6a-f). This conclusion is supported by report by Matsumoto et al. (2015), who observed decreased lamin A/C, SUN1 and SUN2 expression in breast cancer but this decrease seems to be independent of each other [81]. However, SUN2 is significantly inhibited by compressive force. In conclusion, compressive force can decrease SUN2 and lamin A/C levels. Both of these effects compromise cellular mechanotransmission by disrupting the function of the LINC complex. We believe that this negative feedback loop represents a cellular self-protective mechanism involved in the regulation of mechanosensation (Fig. 9).

Compressive force is transmitted through the cytoskeleton to the nuclear envelope, where it affects SUN2 units of the LINC complex and lamin A/C. Deficient lamin A/C increases nuclear permeability, facilitating the translocation of YAP1 and enhancing cellular mechanosensation. Simultaneously, as a counterbalance, the detachment of the LINC complex caused by deficient lamin A/C disrupts intracellular mechanotransmission, inhibiting cellular responses.

Our study highlights the dual roles of lamin A/C in cellular mechanosensation. Compressive force significantly downregulates lamin A/C protein level. This lamin A/C deficiency enhances force-induced-inflammatory genes transcription and proliferative impairment by increasing nuclear permeability and promoting the translocation of YAP1. On the other hand, it also reduces force-induced IRF4 expression and DNA damage by disrupting LINC complex’s connection to nuclei. These findings carry broader implications for understanding the mechanobiology of various cell types and tissues. Future research will help to unravel the relevance of these observations under physiological and pathological conditions.

Acknowledgement

We sincerely thank Dr. Klaus Deckmann and Mrs. Dingding Ai (Department of Anatomy, Justus-Liebig University of Giessen, Germany) for their help on confocal laser scanning microscopy.

We also thank the China Scholarship Council (CSC) for Ph.D. financial support.

Availability of data

All data generated in study appear in the submitted article.

Competing interests

The authors declare that they have no competing interests.

Author Contribution

Yao Wang: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Methodology (equal); Software (equal); Validation (equal); Writing-original draft (equal). Sabine Ruf: Investigation (equal); Methodology (equal); Project administration (equal); Validation (equal); Writing-review & editing (equal). Lei Wang: Formal analysis (equal); Methodology (equal). Sabine Groeger: Formal analysis (equal); Funding acquisition (equal); Methodology (equal); Project administration (equal); Supervision (equal); Validation (equal); Writing-review & editing (equal).

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The Dual Roles of Lamin A/C in Mechanosensation to Compressive Force

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Abstract

Figures

Introduction

1. Method and Materials

1.1 Cell Culture and Compressive Force Loading

1.2 Small interfering RNA oligonucleotides

1.3 Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)

1.4 Proteins Extraction and Western Blot Analysis

1.5 Comet assay

1.6 Evaluation of Nuclear Permeability

1.7 Immunofluorescence (IF) Staining

1.8 Statistical Analysis

2. Results

2.1 Lamin A/C protein was down-regulated by Compressive Force

2.2 Lamin A/C Deficiency enhanced Effects of Compressive Force on proliferation and inflammatory gene expression

2.3 Lamin A/C Deficiency Inhibited Compressive Force-Induced DNA Damage and IRF4 Expression

2.4 Lamin A/C Deficiency Increased Nuclear Permeability to YAP1

2.5 Lamin A/C Deficiency Detached LINC Complex from Nucleus

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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