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Abnormal nuclear shape and impaired mechanotransduction in emerin-deficient cells.

Lammerding J, Hsiao J, Schulze PC, Kozlov S, Stewart CL, Lee RT - J. Cell Biol. (2005)

Bottom Line: Clin.Invest. 113:370-378).Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115, USA. jlammerding@rics.bwh.harvard.edu

ABSTRACT
Emery-Dreifuss muscular dystrophy can be caused by mutations in the nuclear envelope proteins lamin A/C and emerin. We recently demonstrated that A-type lamin-deficient cells have impaired nuclear mechanics and altered mechanotransduction, suggesting two potential disease mechanisms (Lammerding, J., P.C. Schulze, T. Takahashi, S. Kozlov, T. Sullivan, R.D. Kamm, C.L. Stewart, and R.T. Lee. 2004. J. Clin. Invest. 113:370-378). Here, we examined the function of emerin on nuclear mechanics and strain-induced signaling. Emerin-deficient mouse embryo fibroblasts have abnormal nuclear shape, but in contrast to A-type lamin-deficient cells, exhibit nuclear deformations comparable to wild-type cells in cellular strain experiments, and the integrity of emerin-deficient nuclear envelopes appeared normal in a nuclear microinjection assay. Interestingly, expression of mechanosensitive genes in response to mechanical strain was impaired in emerin-deficient cells, and prolonged mechanical stimulation increased apoptosis in emerin-deficient cells. Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.

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Emerin and A-type lamin-deficient cells have impaired mechanotransduction. (a) Expression of the mechanosensitive genes iex-1 and egr-1 in response to mechanical strain is drastically reduced in emerin and A-type lamin-deficient fibroblasts compared with wild-type cells. Expression of the nonmechanically inducible gene gapdh is not significantly altered. The elevated baseline expression of iex-1 and egr-1 seen in the emerin-deficient cells in this Northern blot are not representative, and real-time PCR analysis didn't reveal any significant differences in baseline expression between cell types. (b) Real-time PCR analysis confirms the impaired induction of iex-1 in response to strain in emerin and A-type lamin-deficient cells. Results are normalized to β-tubulin expression and presented as percent induction of baseline levels (induction at 2 h: 270 ± 87.1%, 143 ± 41.4%, and 104 ± 35.9% for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.05 for wild-type vs. A-type lamin-deficient; induction at 4 h: 244 ± 54.8%, 95 ± 20.3%, and 116 ± 39.8% for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively, P < 0.05 for wild-type vs. emerin-deficient and wild-type vs. A-type lamin-deficient cells; differences in baseline expression were not statistically significant between any cell type).
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fig6: Emerin and A-type lamin-deficient cells have impaired mechanotransduction. (a) Expression of the mechanosensitive genes iex-1 and egr-1 in response to mechanical strain is drastically reduced in emerin and A-type lamin-deficient fibroblasts compared with wild-type cells. Expression of the nonmechanically inducible gene gapdh is not significantly altered. The elevated baseline expression of iex-1 and egr-1 seen in the emerin-deficient cells in this Northern blot are not representative, and real-time PCR analysis didn't reveal any significant differences in baseline expression between cell types. (b) Real-time PCR analysis confirms the impaired induction of iex-1 in response to strain in emerin and A-type lamin-deficient cells. Results are normalized to β-tubulin expression and presented as percent induction of baseline levels (induction at 2 h: 270 ± 87.1%, 143 ± 41.4%, and 104 ± 35.9% for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.05 for wild-type vs. A-type lamin-deficient; induction at 4 h: 244 ± 54.8%, 95 ± 20.3%, and 116 ± 39.8% for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively, P < 0.05 for wild-type vs. emerin-deficient and wild-type vs. A-type lamin-deficient cells; differences in baseline expression were not statistically significant between any cell type).

Mentions: Impaired cellular signaling in response to mechanical stimulation (mechanotransduction) can lead to an altered physiological response and to potentially increased apoptosis in mechanically strained tissue. A-type lamin-deficient cells have impaired mechanotransduction signaling in vivo and in vitro (Lammerding et al., 2004; Nikolova et al., 2004), and insufficient anti-apoptotic signaling could provide one explanation for the increased apoptotic cell fractions in A-type lamin and emerin-deficient cells seen in the 24-h strain experiments. In wild-type cells, expression of the mechanosensitive gene egr-1 and the anti-apoptotic gene iex-1 is rapidly up-regulated in response to mechanical stimulation (Sadoshima et al., 1992; Morawietz et al., 1999; De Keulenaer et al., 2002). To evaluate whether transcriptional activation for these genes was altered in A-type lamin or emerin-deficient cells, fibroblasts plated on fibronectin-coated silicone membranes were subjected to biaxial cyclic strain (4%, 1 Hz) and mRNA levels were subsequently analyzed using Northern analysis and real-time PCR. We found that, in addition to the previously established mechanotransduction deficiency in A-type lamin-deficient cells, emerin-deficient cells also exhibited an attenuated mechanotransduction response. Expression of egr-1 and iex-1 in response to mechanical stimulation was impaired in both A-type lamin and emerin-deficient cells at 2 and 4 h of 4% biaxial cyclic strain application (Fig. 6 a). In contrast, the mechanically unresponsive gene glyceraldehyde 3-phosphate dehydrogenase (gapdh) showed no changes in expression in any cell types, suggesting that transcription was impaired in a specific manner. Impaired mechanosensitive expression of iex-1 in A-type lamin and emerin-deficient cells was confirmed by real-time PCR (Fig. 6 b). Expression levels of iex-1 at baseline (nonstimulated cells) were not significantly different between cell types.


Abnormal nuclear shape and impaired mechanotransduction in emerin-deficient cells.

Lammerding J, Hsiao J, Schulze PC, Kozlov S, Stewart CL, Lee RT - J. Cell Biol. (2005)

Emerin and A-type lamin-deficient cells have impaired mechanotransduction. (a) Expression of the mechanosensitive genes iex-1 and egr-1 in response to mechanical strain is drastically reduced in emerin and A-type lamin-deficient fibroblasts compared with wild-type cells. Expression of the nonmechanically inducible gene gapdh is not significantly altered. The elevated baseline expression of iex-1 and egr-1 seen in the emerin-deficient cells in this Northern blot are not representative, and real-time PCR analysis didn't reveal any significant differences in baseline expression between cell types. (b) Real-time PCR analysis confirms the impaired induction of iex-1 in response to strain in emerin and A-type lamin-deficient cells. Results are normalized to β-tubulin expression and presented as percent induction of baseline levels (induction at 2 h: 270 ± 87.1%, 143 ± 41.4%, and 104 ± 35.9% for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.05 for wild-type vs. A-type lamin-deficient; induction at 4 h: 244 ± 54.8%, 95 ± 20.3%, and 116 ± 39.8% for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively, P < 0.05 for wild-type vs. emerin-deficient and wild-type vs. A-type lamin-deficient cells; differences in baseline expression were not statistically significant between any cell type).
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fig6: Emerin and A-type lamin-deficient cells have impaired mechanotransduction. (a) Expression of the mechanosensitive genes iex-1 and egr-1 in response to mechanical strain is drastically reduced in emerin and A-type lamin-deficient fibroblasts compared with wild-type cells. Expression of the nonmechanically inducible gene gapdh is not significantly altered. The elevated baseline expression of iex-1 and egr-1 seen in the emerin-deficient cells in this Northern blot are not representative, and real-time PCR analysis didn't reveal any significant differences in baseline expression between cell types. (b) Real-time PCR analysis confirms the impaired induction of iex-1 in response to strain in emerin and A-type lamin-deficient cells. Results are normalized to β-tubulin expression and presented as percent induction of baseline levels (induction at 2 h: 270 ± 87.1%, 143 ± 41.4%, and 104 ± 35.9% for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.05 for wild-type vs. A-type lamin-deficient; induction at 4 h: 244 ± 54.8%, 95 ± 20.3%, and 116 ± 39.8% for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively, P < 0.05 for wild-type vs. emerin-deficient and wild-type vs. A-type lamin-deficient cells; differences in baseline expression were not statistically significant between any cell type).
Mentions: Impaired cellular signaling in response to mechanical stimulation (mechanotransduction) can lead to an altered physiological response and to potentially increased apoptosis in mechanically strained tissue. A-type lamin-deficient cells have impaired mechanotransduction signaling in vivo and in vitro (Lammerding et al., 2004; Nikolova et al., 2004), and insufficient anti-apoptotic signaling could provide one explanation for the increased apoptotic cell fractions in A-type lamin and emerin-deficient cells seen in the 24-h strain experiments. In wild-type cells, expression of the mechanosensitive gene egr-1 and the anti-apoptotic gene iex-1 is rapidly up-regulated in response to mechanical stimulation (Sadoshima et al., 1992; Morawietz et al., 1999; De Keulenaer et al., 2002). To evaluate whether transcriptional activation for these genes was altered in A-type lamin or emerin-deficient cells, fibroblasts plated on fibronectin-coated silicone membranes were subjected to biaxial cyclic strain (4%, 1 Hz) and mRNA levels were subsequently analyzed using Northern analysis and real-time PCR. We found that, in addition to the previously established mechanotransduction deficiency in A-type lamin-deficient cells, emerin-deficient cells also exhibited an attenuated mechanotransduction response. Expression of egr-1 and iex-1 in response to mechanical stimulation was impaired in both A-type lamin and emerin-deficient cells at 2 and 4 h of 4% biaxial cyclic strain application (Fig. 6 a). In contrast, the mechanically unresponsive gene glyceraldehyde 3-phosphate dehydrogenase (gapdh) showed no changes in expression in any cell types, suggesting that transcription was impaired in a specific manner. Impaired mechanosensitive expression of iex-1 in A-type lamin and emerin-deficient cells was confirmed by real-time PCR (Fig. 6 b). Expression levels of iex-1 at baseline (nonstimulated cells) were not significantly different between cell types.

Bottom Line: Clin.Invest. 113:370-378).Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115, USA. jlammerding@rics.bwh.harvard.edu

ABSTRACT
Emery-Dreifuss muscular dystrophy can be caused by mutations in the nuclear envelope proteins lamin A/C and emerin. We recently demonstrated that A-type lamin-deficient cells have impaired nuclear mechanics and altered mechanotransduction, suggesting two potential disease mechanisms (Lammerding, J., P.C. Schulze, T. Takahashi, S. Kozlov, T. Sullivan, R.D. Kamm, C.L. Stewart, and R.T. Lee. 2004. J. Clin. Invest. 113:370-378). Here, we examined the function of emerin on nuclear mechanics and strain-induced signaling. Emerin-deficient mouse embryo fibroblasts have abnormal nuclear shape, but in contrast to A-type lamin-deficient cells, exhibit nuclear deformations comparable to wild-type cells in cellular strain experiments, and the integrity of emerin-deficient nuclear envelopes appeared normal in a nuclear microinjection assay. Interestingly, expression of mechanosensitive genes in response to mechanical strain was impaired in emerin-deficient cells, and prolonged mechanical stimulation increased apoptosis in emerin-deficient cells. Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.

Show MeSH
Related in: MedlinePlus