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Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2.

Tamplenizza M, Lenardi C, Maffioli E, Nonnis S, Negri A, Forti S, Sogne E, De Astis S, Matteoli M, Schulte C, Milani P, Tedeschi G - J Nanobiotechnology (2013)

Bottom Line: Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF.We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIMAINA and Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, Milano 20133, Italy. paolo.milani@mi.infn.it.

ABSTRACT

Background: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified.

Results: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.

Conclusion: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

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Effect of nanotopography on FAK expression and actin cytoskeleton rearrangement. (A) FAK expression was evaluated by Western blot using anti-FAK antibodies in PC12 cells grown on PLL-coated glass (G-PLL), flat Titania (flat TiO2) and nanostructured Titania (20 nm rms ns-TiO2) in the presence and in the absence of 50 ng/mL NGF. The results are means of 2 different experiments. (B): Stress fiber organization after 48 h of culture in low serum media on different substrates and conditions: G-PLL (A), G-PLL + NGF (B), flat TiO2 (C) and 20 nm rms ns-TiO2 (D). Actin (red) and nucleus (blue) fluorescent staining.
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Figure 3: Effect of nanotopography on FAK expression and actin cytoskeleton rearrangement. (A) FAK expression was evaluated by Western blot using anti-FAK antibodies in PC12 cells grown on PLL-coated glass (G-PLL), flat Titania (flat TiO2) and nanostructured Titania (20 nm rms ns-TiO2) in the presence and in the absence of 50 ng/mL NGF. The results are means of 2 different experiments. (B): Stress fiber organization after 48 h of culture in low serum media on different substrates and conditions: G-PLL (A), G-PLL + NGF (B), flat TiO2 (C) and 20 nm rms ns-TiO2 (D). Actin (red) and nucleus (blue) fluorescent staining.

Mentions: It has been very recently demonstrated that adhesive proteins of the ECM linked with the expression of focal adhesion kinase (FAK), like collagen, fibronectin and laminin, have a profound impact on PC12 cell neurite extension [25]. On the other hand, in PC12 cells grown on biomaterials, such as highly disordered CH3/OH substrates, neuronal adhesion and differentiation mainly depend on nanoscale surface free-energy gradients [32]. To further demonstrate the correlation between nano-topography of TiO2 and cell differentiation, we evaluated FAK expression and actin cytoskeleton rearrangements in PC12 cells cultured on PLL-glass, on ns-TiO2 (20 nm rms) and on flat microcrystalline TiO2. As shown in Figure 3, PC12 cells seeded on ns-TiO2, without NGF treatment, underwent actin cytoskeleton reorganization associated to an increase in FAK expression. As expected, the addition of NGF leads to an increase in FAK expression also in cells seeded on PLL-Glass and on flat-TiO2, while the concomitant presence of two different stimuli (NGF and nano-structure) results in a decrease in FAK expression as compared to cells grown on ns-TiO2 without NGF, an effect that is worth investigating in more details in the future.


Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2.

Tamplenizza M, Lenardi C, Maffioli E, Nonnis S, Negri A, Forti S, Sogne E, De Astis S, Matteoli M, Schulte C, Milani P, Tedeschi G - J Nanobiotechnology (2013)

Effect of nanotopography on FAK expression and actin cytoskeleton rearrangement. (A) FAK expression was evaluated by Western blot using anti-FAK antibodies in PC12 cells grown on PLL-coated glass (G-PLL), flat Titania (flat TiO2) and nanostructured Titania (20 nm rms ns-TiO2) in the presence and in the absence of 50 ng/mL NGF. The results are means of 2 different experiments. (B): Stress fiber organization after 48 h of culture in low serum media on different substrates and conditions: G-PLL (A), G-PLL + NGF (B), flat TiO2 (C) and 20 nm rms ns-TiO2 (D). Actin (red) and nucleus (blue) fluorescent staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3815074&req=5

Figure 3: Effect of nanotopography on FAK expression and actin cytoskeleton rearrangement. (A) FAK expression was evaluated by Western blot using anti-FAK antibodies in PC12 cells grown on PLL-coated glass (G-PLL), flat Titania (flat TiO2) and nanostructured Titania (20 nm rms ns-TiO2) in the presence and in the absence of 50 ng/mL NGF. The results are means of 2 different experiments. (B): Stress fiber organization after 48 h of culture in low serum media on different substrates and conditions: G-PLL (A), G-PLL + NGF (B), flat TiO2 (C) and 20 nm rms ns-TiO2 (D). Actin (red) and nucleus (blue) fluorescent staining.
Mentions: It has been very recently demonstrated that adhesive proteins of the ECM linked with the expression of focal adhesion kinase (FAK), like collagen, fibronectin and laminin, have a profound impact on PC12 cell neurite extension [25]. On the other hand, in PC12 cells grown on biomaterials, such as highly disordered CH3/OH substrates, neuronal adhesion and differentiation mainly depend on nanoscale surface free-energy gradients [32]. To further demonstrate the correlation between nano-topography of TiO2 and cell differentiation, we evaluated FAK expression and actin cytoskeleton rearrangements in PC12 cells cultured on PLL-glass, on ns-TiO2 (20 nm rms) and on flat microcrystalline TiO2. As shown in Figure 3, PC12 cells seeded on ns-TiO2, without NGF treatment, underwent actin cytoskeleton reorganization associated to an increase in FAK expression. As expected, the addition of NGF leads to an increase in FAK expression also in cells seeded on PLL-Glass and on flat-TiO2, while the concomitant presence of two different stimuli (NGF and nano-structure) results in a decrease in FAK expression as compared to cells grown on ns-TiO2 without NGF, an effect that is worth investigating in more details in the future.

Bottom Line: Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF.We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIMAINA and Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, Milano 20133, Italy. paolo.milani@mi.infn.it.

ABSTRACT

Background: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified.

Results: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.

Conclusion: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

Show MeSH
Related in: MedlinePlus