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M. Morales, E. Syriani, A. Gomez-Cabrero, J. Pintor, X. Gasull; Control of Actin Dynamic and IOP by Profilin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):357.
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It is well known that modulation of actin cytoskeleton and cellular contractility in trabecular meshwork cells (TM) regulates outflow facility and intraocular pressure (IOP). Profilin has been implicated in cell motility and in a variety of cellular processes such as membrane extension, endocytosis and formation of focal complexes. To study the role of Profilin regulating TM physiology we have generated a membrane permeable version of profilin I (PTD4-PfnI). Here, we have investigated the role of Profilin regulating actin dynamic and cell morphology in TM cells and its participation in IOP regulation.
Recombinant proteins were expressed in Escherichia coli BL21-PLys bacteria and purified through a Ni-NTA resin. Effects of PTD4-Pfn I on actin cytoskeleton and time lapse microscopy was studied in bovine TM cells culture. Rac 1 activation was assessed by GST-CRIB pull down protocol. Changes in IOP were measured after topical application in rabbit eyes corneas by means of a Tonopen for a period up to 8 hours.
In TM cells profilin I induces cell spreading and lamella formation independently of growth factor presence. The effects are time- and concentration-dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. PTD4-Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS-induced lamellipodia formation activates Rac1, PTD4-Pfn I stimulation did not induce Rac1 activation. Time-lapse microscopy confirmed the effects of profilin on lamellipodia extension with a higher membrane spreading velocity than FBS. In vivo when 10 µM of PTD4- Pfn I was topically applied to rabbits corneas, the IOP was reduces in 20 % compared to the control vehicle. The mean time effect of PTD4-PfnI was 6.6 hours
Profilin I is an important player in TM cell cytoskeleton. Up-regulation of intracellular levels of profilin induces cell membrane spreading in a Rac 1 independent activation pathway. Furthermore, topical-corneal application of PTD4-PfnI was able to reduced intraocular pressure. We propose Profilin as a new pharmacological target for the treatment of ocular hypertension.
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