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3 3 4. Materials and Methods 4 4.1 PISK/AKT signaling pathway activation upon Plant-IGF1 treatment 4 4.2 Effect of Plant-IGF1 on the activation of anti-apoptotic genes 5 5. Results 6 5.1 PISK/AKT signaling pathway is induced upon plant-IGF1 treatment 6 5.2 Plant-IGF1 inhibits apoptosis by the activation of anti-apoptotic genes 7 6. Conclusions 8 7. References 8 Page 2 of 8 1. Abstract Insulin-like growth factor 1 (|GF1) belongs to the insulin-like growth factor family of ‘signaling molecules that play critical roles in cellular energy metabolism and in growth and development. Plant produced IGF1 is active and able to induce keratinocytes and fibroblasts proliferation in a dose-dependent manner (nearly 50% ‘and 12%, respectively). In addition, it induces wound closure of skin cells with a high efficiency early after addition, reaching nearly 80% ot in vitro cicatrization after 48h in culture. Moreover, this insulin-like protein activates anti-apoptotic genes like Bcl-2, suggesting a dual effect tending to favor cell proliferation, where the PISK/AKT signaling pathway would be involved. All together suggests IGF1 would bbe a key molecule involved in skin regeneration and remodeling, 2. Title Analysis of the effects of plant-IGF1 upon skin cells: metabolic pathways. 3. Introduction Insulin-like growth factor 1 (IGF1) is a protein with structural homology to pro-insulin. ‘The major source of circulating IGF1 in postnatal life is the liver but IGF1 can also be Produced by many other tissues, where it is thought to act in 2 paracrine fashion (Haase et al, 2003) IGF1, produced by skin fibroblasts and keratinocytes, promotes collagen expression as well as inhibits the expression of the matrix metalloproteinase-1 (MMP-1), a key involved in the break-down of the collagen matrix (Kilani ef al, 2007; van Drielen ef al, 2015) suggesting IGF1 would be a relevant protein in ageing and wound healing enzyme Wound healing is a complex biological process that requires cellular interactions between a variety of cells, including fibroblasts, myofibroblasts, smooth muscle cells, ‘endothelial cells, keratinocytes and immune cells. These interactions are mediated by numerous factors such as hormones, blood components, second messengers and growth factors like epidermal growth factor (EGF), fibroblast growth factor (FGF) and the insulin-like growth factor (IGF1), among others (GrazulBilska ef al, 2003). The relevance of IGF1 in wound healing has been demonstrated in diabetic individuals Page 3 of 8 where decreased levels of this growth factor were correlated with non-healing skin wounds (Blakytny ef al, 2000). IGF1, a well characterized survival factor, is a potent activator of the PISK/AKT pathway (Zhang et al, 2012). AKT plays a central role in promoting the survival of a wide range of cell types (Dudek ef al, 1997). This pathway has been implicated in the regulation of cell proliferation, transformation, actin cytoeskeletal organization and apoptosis, by activation of different effectors (Toker and Cantley, 1997). The proapoptotic function of the BCL-2 family member Bad and caspase 9 are known to be suppressed by AKT (Brunet ef al, 1999), thus favoring cell survival, PISK/AKT dependent pathway would stimulate epithelialization in an additive manner with the one activated by EGF (Haase et al, 2003), 4. Materials and Methods + Keratinocytes (HaCaT): from CLS (Cell Lines Service) ‘+ Fibroblasts: Primary fibroblasts from biopsy. ‘* Plant-IGF1: produced in Nicotiana benthamiana and purified from leaves. 4.1 PI3K/AKT signaling pathway activation upon plant-IGF1 treatment Procedure: ‘+ Day 1: Fibroblasts were seeded in a 6 wells plate in complete DMEM (10%FBS) until 90% confluence was reached. ‘+ Day 2: medium was changed by fresh DMEM deprived of FBS. Day 3: PlantiGF1, re-suspended according to the QC sheet, was added at 50ng/ml. Basal: Cells maintained in the absence of plant-1GF1 + Day 4: RNA extraction and Westem blot for p-AKT and B-Actin. Densitometry was used to quantify protein bands. Page 4 of 8 42 Effect of plant-IGF1 on the activation of anti-apoptotic genes Procedure: Day 1: Keratinocytes were seeded in a 6 wells plate in complete DMEM (10%FBS) until 90% confluence was reached. Day 2: medium was changed by fresh DMEM deprived of FBS, Day 3: Plant-IGF1 Activation Kinetics: Plant-IGF1, re-suspended according to the QC sheet, was added to each well at SOng/ml and the kinetics was done at 07, 5, 10", 15%, 30° and 45° Basal: Time 0' of the kinetics. Day 4: Protein quantification and Westem biot for the anti-apoptotic protein Bcl- 2. Densitometry was used to quantity protein bands. Page 5 of 8 5 Results 1 PI3K/AKT signaling pathway is induced upon plant-IGF1 treatment AA progressive activation in AKT phosphorylation during the kinetics is detected, where the major differences are observed early in the time curve (a 4 fold increase in the amount of P-protein is detected after 5° plant1GF1 addition) (Fig. 1). ERK phosphorilation was also tested but there were no differences in the level of p-ERK between the basal and the plant1GF1 conditions (not shown). Basal 5 1 30 4s Time (rnin) Figure 1. A: Westem blot protein bands quantification by Densitometry. The intensity of each p- ‘AKT band is normalized by its corresponding B-ACTIN band. B. Fold increase in the amount of phosphorylated AKT protein after piant-IGF' treatment. The value of “1” is arbitrary assigned to the Basal, in order to make the fold increase calculations. The “Basaf’ condition are primary fibroblasts maintained in complete DMEM (with no plantIGF1 addition). PlantiGF1 was added to a set of wells at 5', 15°, 30° and 45° (kinetics). Data are the mean of three independent ‘experiments # the Standard Error (SEM). Page 6 of 8 5.2 Plant-IGF1 inhibits apoptosis by the activation of anti- apoptotic genes ‘There is a progressive increase in the amount of the anti-apoptotic protein BCL-2 during the kinetics, where the major differences are observed early in the time curve (a 1.5 fold increase after 5‘ treatment) (Fig. 2). 5 10 18 304s = 110589796.00 27600317.00 12478711.00 22548075.00 22020832.00 21363640.00 28877539.00 23806589.00 30220882.00 26311368.00 31106075.00_21342246.00 4,00 B. 350 3.00 250 Fold increase B8588 BASAL. 10 15 30 “s ‘Tire (rnin) Figure 2. A: Wester biot protein bands quantification by Densitometry. The intensity of each BCL-2 band in the kinetics is normalized by its corresponding B-ACTIN band. B: Fold increase in the amount of BCL-2 protein after plant-IGF1 treatment. The value of “1” is arbitrary assigned to the Basal, in order to make the fold increase calculations. The “Basal” condition are keratinocytes maintained in complete DMEM (with no plantiGF1 addition). Plant-IGF1 was added to a set of wells at 5°, 10, 15, 30° and 45° (kinetics) and the amount of Bcl2 protein determined Page 7 of 8 6 Conclusions Plant-lGF1 is able to activate anti-apoptotic genes on keratinocytes. Specifically, the amount of BCL-2 is rapidly increased upon plantIGF1 treatment reaching a 2.5 fold increase regarding the basal after 10’ plantGF1 addition. Moreover, plant-IGF1 ‘activates the PISK/AKT pathway early in the activation kinetics, suggesting its role in IGF1 signaling. The dual effect of piant-IGF1 promoting anti-apoptosis as well as induction of skin cells proliferation would support its role in regeneration and remodeling, 7 References Blakytny R., Jude E.B., Gibson J.M., Boulton A.J.M. and Mark W.J. Ferguson. 2000. Lack of insulin-Ike growth factor 1 (IGF1) in the basal keratinocyte layer of diabetic skin and diabetic foot ulcers. The Journal of Pathology 190(5): 589-594 Brunet A., Bonni A., Zigmond M. J., Lin M. Z., Juo P., Hu L.S., Anderson M.J., Arden K.C., John Blenis J., and Michael E. Greenberg. 1999. Akt Promotes Cell Survival by Phosphorylating ‘and inhibiting a Forkhead Transcription Factor. Cell 96: 857-868. Drislen K., Gunn D. A., Noordam R., Griffiths C.E.M., Westendorp R.G.J., de Craen AIM. and van Heemst D. 2015. Disentangling the effects of circulating IGF-1, glucose, and cortisol on features of perceived age. Age 37(3):9771. dol: 10.1007/s11357-015-9771-3. Dudek H., Datta S.R., Franke TF, Brinbaum M., Yao R., Cooper G.M., Segal R.A., Kaplan D.R., and Greenberg MLE, 1997. Regulation of neuronal survival by the serine-threonine protein kinase Akt. Sclence 275; 661-865. GrazulBilska AT., Johnson M.L, Bilski JJ., Redmer DA. Reynolds LP. Abdullah A, ‘Abdullah KM. 2003. Wound healing: The role of growth factors. Drugs Today 39(10): 787-800. Haase |., Evans R., Pofahl R. and Watt F. 2003. Regulation of keratinocyte shape, migration ‘and wound epithelialization by IGF1 and EGF-dependent signaling pathways. J of Cells Science 116: 3227-3238, Ruhangiz T. Kilani, Lary Guilbert, Xiaoyue Lin and Aziz Ghahary. 2007. Keratinocyte Conditioned medium abrogates the modulatory effects of IGF-1 and TGF-81 on collagenase ‘expression in dermal fibroblasts. Wound Repair and Regeneration 15 (2): 236-244. Toker A. and Cantley L. 1997. Signaling through the lipid products of phosphoinositide-3-OH_ kinase, Nature 387: 673-676, Zhang S, Zhang Y, Zhuang Y, Wang J, Ye J, et al. 2012, Matrine Induces Apoptosis in Human Acute Myeloid Leuicemia Cals vin the Miochondial Pathway and Akt Inacivaion, PLOS ONE (10): 648853. Page 8 of 8