HSP25 Is Involved in Two Steps of the Differentiation of PAM212 Keratinocytes

Abstract

HSP25 is a member of the small heat shock protein family. This 25-kDa protein exhibits a highly specific distribution during mouse embryonic development. Although multiple functions have been proposed for HSP25, the role it plays during differentiation is still unknown. High levels of HSP25 can be detected in embryonic and adult skin. During epidermis differentiation, the concentration of HSP25 increases with the distance of keratinocytes from the basal layer, in parallel with the extent of keratinization. We used an ex vivo cellular system, PAM212 cells, to analyze quantitatively and qualitatively the dynamics of HSP25 production and phosphorylation during the differentiation of keratinocytes. Our observations suggest that HSP25 is involved in two steps of PAM212 keratinocyte differentiation. Shortly after the induction of differentiation, a transient hyperphosphorylation of HSP25 seems to be essential for the expression of differentiation markers. Later, the chaperone-active form of HSP25 is organized progressively into characteristic aggregates involved in the dynamics of keratin filament networks. HSP25 is a member of the small heat shock protein family. This 25-kDa protein exhibits a highly specific distribution during mouse embryonic development. Although multiple functions have been proposed for HSP25, the role it plays during differentiation is still unknown. High levels of HSP25 can be detected in embryonic and adult skin. During epidermis differentiation, the concentration of HSP25 increases with the distance of keratinocytes from the basal layer, in parallel with the extent of keratinization. We used an ex vivo cellular system, PAM212 cells, to analyze quantitatively and qualitatively the dynamics of HSP25 production and phosphorylation during the differentiation of keratinocytes. Our observations suggest that HSP25 is involved in two steps of PAM212 keratinocyte differentiation. Shortly after the induction of differentiation, a transient hyperphosphorylation of HSP25 seems to be essential for the expression of differentiation markers. Later, the chaperone-active form of HSP25 is organized progressively into characteristic aggregates involved in the dynamics of keratin filament networks. HSP25 (called HSP27 in human cells) belongs to the small heat shock protein (HSP) 1The abbreviations used are: HSP, heat shock protein; BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; MAPKAP, MAP kinase-activated protein; MEK, MAP/ERK kinase; MEKK, MEK kinase; MK, mouse keratin; PBS, phosphate-buffered saline. family. All members of this family are small (20–30 kDa) and contain a highly conserved carboxyl-terminal region, called the α-crystallin domain (1Hickey E. Brandon S.E. Potter R. Stein G. Stein J. Weber L.A. Nucleic Acids Res. 1986; 14: 4127-4145Crossref PubMed Scopus (195) Google Scholar). HSP25 was the first of the 10 mammalian members of this family to be described. Its synthesis is strongly up-regulated in response to heat and chemical and oxidative stresses. Two residues from HSP25 can be phosphorylated via the p38 MAP kinase pathway: Ser-15 and Ser-86. p38 phosphorylates MAPKAP kinase 2/3 which in turn phosphorylates HSP25 (2Stokoe D. Engel K. Campbell D.G. Cohen P. Gaestel M. FEBS Lett. 1992; 313: 307-313Crossref PubMed Scopus (472) Google Scholar). αA- and αB-crystallin were initially described as eye lens structural proteins. However, αB-crystallin has a function and sequence similar to those of HSP25. It can be induced by heat and chemical and oxidative stresses. These proteins are well known chaperones (3Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 4Ehrnsperger M. Gaestel M. Buchner J. Methods Mol. Biol. 2000; 99: 421-429PubMed Google Scholar), although no substrate specificity has been described so far. During embryonic development, HSP25 appears to be strongly produced in specific organs or tissues such as heart, skeletal muscles, smooth muscles, epidermis, eye lens, cartilage, and bone (5Gernold M. Knauf U. Gaestel M. Stahl J. Kloetzel P.M. Dev. Genet. 1993; 14: 103-111Crossref PubMed Scopus (106) Google Scholar, 6Loones M.T. Rallu M. Mezger V. Morange M. Cell. Mol. Life Sci. 1997; 53: 179-190Crossref PubMed Scopus (58) Google Scholar, 7Morange M. Favet N. Loones M.T. Manuel M. Mezger V. Michel E. Rallu M. Sage J. Ann. N. Y. Acad. Sci. 1998; 851: 117-122Crossref PubMed Scopus (10) Google Scholar, 8Morange M. Bukau B. Molecular Chaperones and Folding Catalysts: Regulation, Cellular Function, and Mechanisms. Harwood Academic Publishers, Amsterdam1999: 163-176Google Scholar, 9Davidson S.M. Loones M.T. Duverger O. Morange M. Prog. Mol. Subcell. Biol. 2002; 28: 103-128Crossref PubMed Scopus (22) Google Scholar). Immunohistochemical analyses have detected HSP25 in some areas of the developing central nervous system, even though global Western and Northern blotting failed to detect this protein (10Loones M.-T. Chang Y. Morange M. Cell Stress Chaperones. 2000; 5: 291-305Crossref PubMed Scopus (66) Google Scholar, 11Armstrong C.L. Krueger-Naug A.M. Currie R.W. Hawkes R. J. Comp. Neurol. 2001; 434: 262-274Crossref PubMed Scopus (47) Google Scholar). This specific pattern suggests that this small heat shock protein is involved in cell differentiation, which is further supported by the results obtained by a few cellular approaches. The ex vivo differentiation of HL-60 promyelocytic leukemic cells into macrophages or granulocytes is accompanied by the transient phosphorylation of HSP27 and by an increase in the production of the protein and a decrease in proliferation (12Spector N.L. Ryan C. Samson W. Levine H. Nadler L.M. Arrigo A.P. J. Cell. Physiol. 1993; 156: 619-625Crossref PubMed Scopus (59) Google Scholar, 13Spector N.L. Mehlen P. Ryan C. Hardy L. Samson W. Levine H. Nadler L.M. Fabre N. Arrigo A.P. FEBS Lett. 1994; 337: 184-188Crossref PubMed Scopus (41) Google Scholar). The use of antisense oligonucleotides to reduce the production of HSP27 in HL-60 cells leads to a less pronounced reduction in cell growth after induction with retinoic acid and alters some parameters of granulocytic differentiation (14Chaufour S. Mehlen P. Arrigo A.P. Cell Stress Chaperones. 1996; 1: 225-235Crossref PubMed Scopus (41) Google Scholar). However, HSP25 phosphorylation is not essential for HL-60 cell differentiation (15Schultz H. Rogalla T. Engel K. Lee J.C. Gaestel M. Cell Stress Chaperones. 1997; 2: 41-49Crossref PubMed Scopus (10) Google Scholar). During the pluripotent differentiation of embryonic stem cells, HSP27 is dephosphorylated rapidly after induction, which precedes the transient accumulation and oligomerization of the protein. In these cells, the overexpression of HSP27 reduces the rate of cell proliferation, whereas the down-regulation of the protein by stable antisense RNA compromises the differentiation program, causing massive cell apoptosis (16Mehlen P. Mehlen A. Godet J. Arrigo A.P. J. Biol. Chem. 1997; 272: 31657-31665Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). HSP27 as a differentiation and The ex vivo differentiation of is accompanied by a transient increase in HSP27 down-regulation of HSP27 leads to the of differentiation and a massive to cell whereas the overexpression of this protein strongly the of HSP27 is a in the of cell during differentiation P. V. V. C. L. Arrigo A.P. Cell PubMed Scopus Google Scholar). The production of HSP25 and the of p38 MAP kinase are essential for the differentiation of cells into However, the of p38 MAP kinase to be of HSP25 accumulation S.M. Morange M. Dev. Biol. 2000; PubMed Scopus Google Scholar). The overexpression of HSP25 in cells, which are to into that HSP25 levels decrease the cellular of cells and with differentiation into N. Duverger O. Loones M.T. A. O. Morange M. Cell 2001; PubMed Scopus (22) Google Scholar). In and cell for HSP25. HSP25 can with and the of G. J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, T. M. B. J. PubMed Scopus Google Scholar, R. K. S. M. J. G. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). HSP25 phosphorylation the p38 kinase is for the of and filament dynamics E. Weber L.A. J. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, H. E. Weber L.A. J. Mol. Cell. Biol. PubMed Scopus Google Scholar, J. H. G. J. J. J. Cell Sci. 1997; PubMed Google Scholar, H. J. Cell. Physiol. 1998; PubMed Scopus Google Scholar, C. S.E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. J. 1997; PubMed Scopus Google Scholar). It with of the small and αB-crystallin in T. A. Cell. Full Text PDF PubMed Scopus Google Scholar, J. G. M. 1992; PubMed Scopus Google Scholar, S. A. T. E. 1992; Google Scholar, A. Cell Mol. 1993; PubMed Scopus Google or J. 1994; PubMed Scopus Google However, HSP27 is involved in such T. A. J. Y. J. 1993; Google Scholar, L. A. A.M. J. Cell Sci. PubMed Google Scholar). HSP25 can apoptosis C. A. A. Arrigo A.P. E. J. PubMed Scopus Google Scholar, C. P. L. C. S. Arrigo A.P. G. E. C. Cell Biol. 2000; 2: PubMed Scopus Google Scholar, H. J. Mol. Cell. Biol. 2000; PubMed Scopus Google and with R. G. Dev. 2000; 14: Google Scholar). In this the role of HSP25 in the differentiation of mouse In the adult the epidermis of cell The of differentiation increases from the to the of the basal layer, layer, layer, and the The cells in the basal mouse and whereas those in the the and 10 HSP27 can be detected in the human epidermis from of HSP27 increases with the distance of the keratinocytes from the basal layer, in parallel with the extent of C. D. G. M. J. 1998; PubMed Scopus Google Scholar). In human HSP27 with and of the layer, that it plays a role in the of keratin C. G. C. D. H. J. 2002; PubMed Scopus Google Scholar). We used PAM212 cells as a for keratinocyte differentiation. This mouse cell can in a with a and differentiation of these keratinocytes can be induced by the concentration of the P. B. Res. Google Scholar). The pattern of keratin synthesis in these cells is similar to that in the epidermis, whereas of mouse epidermis to ex vivo the P. J. Full Text PDF PubMed Google Scholar). the PAM212 cell is an for the role of HSP25 in the differentiation of the We that HSP25 is involved in two of PAM212 keratinocyte differentiation. a transient accumulation of the phosphorylated form of HSP25 seems to be essential for the induction of differentiation. the chaperone-active form of HSP25 to the of the keratin by the of the Cell and cells were in in in with and differentiation, PAM212 cells were and in the High was p38 10 was to the and were by cells in PBS, into with and for 10 PubMed Scopus Google Scholar). concentration was the protein and of protein were an from were in were was the The were and HSP25 The was of protein of HSP25, with and with were by in and in The was with and the with 10 were to were from the in the of a and to in for were HSP25 was detected by as described were and to were with and in for were in and in The cells were to in a the cells, were in in for in in for cells were with in and for The were in and in and with and for in the in PBS, were with and a or a The were HSP25 from and The were and analyses were in in were in a were in and were of in two of an and in for was in a were with in for were for in a in for were for 10 in BSA, in with in for in were with in for were in and in were a was the of an from a of by The were used as for cells were with and acid in for in the in for and with of a in in a of was the cells and for and for of cells in were and in were and with and and in a The of PAM212 during keratinocytes were in differentiation was induced by the concentration of in the The of the cells was by in the cells, induction were and PAM212 cells were were the cells the cells were organized into two still be cells the cells in the a of cells, it to cell were these and The of PAM212 cells be after with were well from that is of cells this the and some that cells to form a The of the to increase with such that and the of cell of cell was with these or cell were The to be the cells that cells The of HSP25 during the of PAM212 used Western blotting to analyze the expression of specific differentiation to the differentiation of the keratinocytes and are the two produced by the of the epidermis in In PAM212 cells, be detected as as and during the However, not be detected and The of HSP25 during the differentiation Western detected a of HSP25 in cells in a and During differentiation in the of HSP25 The of HSP25 These results that PAM212 cells are a for differentiation and that this is for the role of HSP25 in the differentiation of keratinocytes. HSP25 during the of the used to analyze the distribution of HSP25 in PAM212 cells and during differentiation. were in the to the cell of contain in cell and for HSP25 and We that HSP25 aggregates progressively during the differentiation of PAM212 cells and induction, the protein was in the a and and the cells a and The of HSP25 and not these and the cells and those in the a of the cell in a HSP25 was no in these cells the distribution of was characteristic in the cells in the the and In these cells, HSP25 to form These were the cell and the and The HSP25 and to the and the in cells were the the the cells, and were still the These cells were and to the a This pattern of not during the steps of differentiation and the HSP25 to in some of the to be of HSP25 of HSP25 were detected the of these HSP25 aggregates in the and in the the aggregates were the in a of the cells were the the and in the and these aggregates were in These This characteristic pattern suggests that HSP25 is the of areas that not contain HSP25 and that or not contain These aggregates and not in of the cell and HSP25 was in the of some cells of HSP25 and during the differentiation of PAM212 PAM212 cells were as in and HSP25 and and and were detected by and of the All observations were a and a cell to and the The in and an of an HSP25 progressively aggregates that to the of the cells and in the This highly the increase in the of strongly that HSP25 plays a specific role in the differentiation of PAM212 HSP25 in the of during PAM212 Cell the differentiation of the keratin and in basal cells in is by a keratin and in cells in We have that and are induced during the differentiation of PAM212 that HSP25 with in HSP25 is involved in this the pattern of HSP25 with that of of these We the pattern of HSP25 with that of which is in PAM212 cells and a of the and described HSP25 was in the these a and The of HSP25 and not to these and the cells in the no HSP25 or The distribution of and HSP25 in the cells from this similar aggregates The aggregates of HSP25 and are or that exhibits the pattern as and HSP25 not HSP25 with the in the basal in HSP25 seems to be involved in the of the keratin during the differentiation of PAM212 and were not in PAM212 cells induction during differentiation. not and HSP25 in the expression of and the pattern of which is similar to that of HSP25 from was in cells that to cells and these cells no The cells in the were and which it to the the of a of from the that the cells were from those The was a of the cells and were still with cells or not We the expression of and cells and some cells These observations are with the of differentiation expression by Western is initially induced in cells HSP25 that has to cells no contain HSP25. HSP25 and to in the of described HSP25 and was these two proteins are not produced in the cells in adult is specific for the basal layer, whereas HSP25 is in the We these two proteins be detected in the cells during epidermis development. by the expression of HSP25 and in mouse epidermis of the and after the of We that HSP25 and are in the the epidermis the epidermis is and HSP25 was in the known to and whereas the basal not contain HSP25 HSP25 and such as those in PAM212 cells, are to the epidermis a and the it is during embryonic development. HSP25 a after and the of p38 MAP the increase in the HSP25 during the differentiation of PAM212 cells and highly pattern were accompanied by a in the phosphorylation of the protein. The phosphorylation of HSP25 is known to be the p38 MAP kinase p38 phosphorylates MAPKAP kinase which phosphorylates HSP25. p38 can be by the We the of this the differentiation of PAM212 cells was to the or In cells were and proteins were and The of or the differentiation of PAM212 cells, as by the of or not the expression of that PAM212 cells in these that is in the expression of in cells with from The expression of in cells was strongly and with that of in cells in the These results suggest that p38 is in the differentiation and that this is essential for the expression of and that was detected p38 be involved this the of the expression of this was 10 after induction and strongly from that the expression of and is the of p38 a few after induction, the dynamics of HSP25 phosphorylation during the after HSP25 was phosphorylated phosphorylated induction and after induction, two phosphorylated of HSP25 the of which and after induction the two phosphorylated were still although in a whereas the phosphorylated no be detected in these not p38 is essential for the differentiation of PAM212 cells, and HSP25 is a few after induction, in of p38 MAP the of used to analyze PAM212 cells with from to HSP25 aggregates p38 is in the of PAM212 cells were and the of the was similar to that in the cells in the in the cells in the was the and in the and However, the pattern of HSP25 was strongly by the of to was in the cells in the still HSP25 and those in the not the aggregates described The pattern of HSP25 that with the of the cell and The of HSP25 and not and In the the distribution of was the as that of HSP25 from the Western was in a few cells in the less those in and the of p38 MAP kinase not differentiation, alters the pattern of HSP25. HSP25 and the of that aggregates HSP25, and form progressively during the differentiation of PAM212 These observations that HSP25 with the two the keratin in these cells have small and The first of such from the of the of characteristic cellular protein with αB-crystallin T. A. Cell. Full Text PDF PubMed Scopus Google HSP27 T. A. J. Y. J. 1993; Google Scholar). were to be by the of αB-crystallin with J. G. M. 1992; PubMed Scopus Google Scholar, S. A. T. E. 1992; Google Scholar, A. Cell Mol. 1993; PubMed Scopus Google Scholar). of a eye lens that and αB-crystallin with and J. 1994; PubMed Scopus Google Scholar). The that αB-crystallin can in HSP27 was to with protein and αB-crystallin in cells small and to with keratin in cells that not contain αB-crystallin L. A. A.M. J. Cell Sci. PubMed Google Scholar). to these the pattern of the small the filament though have been the of this has not been The that a in αB-crystallin filament in P. A. P. A. D. D. M. Genet. 1998; PubMed Scopus Google of a role for small with We not the pattern of HSP25 and the keratin induction of PAM212 The and from initially and involved in the of characteristic These aggregates are from those small and in This is the first that such HSP25 and have been described. an keratin and HSP27 in cells L. A. A.M. J. Cell Sci. PubMed Google Scholar), these cells a stable and well keratin The of be of the differentiation of a from a keratin to a Our observations suggest that HSP25 the of the keratin the of keratin and that is initially in cells for aggregates to of cell that not contain these it is that the keratin of cells has to be the keratin of cells can be The the of and the expression of and is by the that differentiation and However, it is not the of the is a of differentiation induction or it differentiation. in PAM212 cells to and HSP25 not with which is an filament of the as However, in cells in with HSP27 with protein the expression of which was by L. A. A.M. J. Cell Sci. PubMed Google Scholar). This further the specificity of the and in this and of HSP25 to the dynamics of HSP25 phosphorylation differentiation, the the form of HSP25, which form known to be with the of HSP25 T. M. A. G. C. C. M. Arrigo A.P. Buchner J. Gaestel M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that the of HSP27 is during the keratinocyte differentiation of human cells and that this HSP27 in the form of A.P. C. 2002; PubMed Scopus Google Scholar). However, the the accumulation of the chaperone-active form of HSP27 to be a of and that it was not to be to a differentiation Our results that during PAM212 cells differentiation HSP25 with proteins that are to the differentiation The chaperone-active form of HSP25 with proteins. the of the a of keratin that are by HSP25 to and and are the first of the HSP25 described so far. This is the first that HSP25 is involved in a specific differentiation with specific However, these not the of the We that the of aggregates be a first by the this is not in with the of the the of cells a the cells in which these suggests that can still be used to an This is in with the that the with the chaperone-active form of HSP25 are not can M. S. Gaestel M. Buchner J. J. 1997; PubMed Scopus Google Scholar). The of the and the of to be HSP25 p38 and of PAM212 is a few after induction of PAM212 cell differentiation. the phosphorylation of HSP25 and after The two phosphorylated were still detected after induction, were the The phosphorylation of HSP25 is the p38 MAP kinase (2Stokoe D. Engel K. Campbell D.G. Cohen P. Gaestel M. FEBS Lett. 1992; 313: 307-313Crossref PubMed Scopus (472) Google Scholar). The role of MAP in keratinocyte differentiation has been in human differentiation induced with a MAP kinase protein kinase p38 and protein T. P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In system, the induction of differentiation after an increase in concentration was to be of protein kinase and and to the of and MEK, the transient of ERK, and an increase in with cell However, in this system, was not to be to differentiation, that in with the M. M. G. S.M. S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that p38 MAP kinase is in the differentiation of PAM212 These that HSP25 is a few after induction and that the of p38 MAP kinase is essential for differentiation. the phosphorylation of HSP25 be involved in the differentiation such as J. Y. 1997; PubMed Scopus Google Scholar, D. 1996; 272: PubMed Scopus Google or protein R. T. J. 1997; PubMed Scopus Google Scholar, A. J. 1997; PubMed Scopus Google Scholar, M. L.M. J. 1998; PubMed Scopus Google be by the of the that the phosphorylation of HSP25 is rapidly by the induction of the that HSP25 phosphorylation is involved in this induction in with HSP25 phosphorylation R. K. S. M. J. G. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, T. K. J. M. B. J. Cell Biol. PubMed Scopus Google in a that differentiation. This is to be the of dynamics is essential for V. C. M. E. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). HSP25 to with the of the dynamics of HSP25 during the steps of the differentiation a of during these PAM212 and expression of the differentiation that further PAM212 cells as a for epidermis differentiation. During differentiation, PAM212 cells which is as the of cells of a are of a and a the contain that PAM212 cells a parallel this pattern of and the of the cell in the cells the basal and cells the In even though PAM212 cells the induction, the cells no or that not to basal or These cells as a for the keratinocytes involved in the differentiation from the of the was the of the distribution of HSP25 in the of the and the distribution of HSP25 in and PAM212 the in basal cells not contain HSP25, whereas cells contain levels of HSP25. the that HSP25 is in PAM212 cells and with these and that it is in cells it is to that the observations in PAM212 cells to a in a it is of The of the expression of HSP25 and in developing mouse epidermis that the two proteins are in the cells in the epidermis, the HSP25 and in PAM212 cells is to to a the of epidermis and not a the epidermis of cell These observations suggest that with PAM212 cells is not a of in or adult epidermis, which is involved in a this the of during embryonic development. These results that HSP25 has in the of the epidermis in and adult it with HSP25 and Cellular HL-60 promyelocytic leukemic cell differentiation have that cell proliferation as HSP27 levels increase (12Spector N.L. Ryan C. Samson W. Levine H. Nadler L.M. Arrigo A.P. J. Cell. Physiol. 1993; 156: 619-625Crossref PubMed Scopus (59) Google Scholar, 13Spector N.L. Mehlen P. Ryan C. Hardy L. Samson W. Levine H. Nadler L.M. Fabre N. Arrigo A.P. FEBS Lett. 1994; 337: 184-188Crossref PubMed Scopus (41) Google and that HSP27 is essential for the differentiation of HL-60 cells (14Chaufour S. Mehlen P. Arrigo A.P. Cell Stress Chaperones. 1996; 1: 225-235Crossref PubMed Scopus (41) Google Scholar). the pluripotent differentiation of embryonic stem cells (16Mehlen P. Mehlen A. Godet J. Arrigo A.P. J. Biol. Chem. 1997; 272: 31657-31665Abstract Full Text Full Text PDF PubMed Scopus (197) Google and differentiation P. V. V. C. L. Arrigo A.P. Cell PubMed Scopus Google have that HSP27 as a differentiation and have the of HSP25 in cell differentiation, the function of the protein in the from involved in the cell proliferation, and differentiation, the specific role of HSP25 in the organs the protein is produced in vivo unknown. that HSP25 the of the during PAM212 keratinocytes differentiation. This is specific to cell differentiation. that HSP25 has functions in cell differentiation to the This protein with specific in HSP25 during development. in vivo observations suggest that HSP25 is involved in the differentiation of a of not in differentiation It is to the role of HSP25 in cell differentiation. We are to Y. of for the of the system, S. of for the PAM212 cells, M. for the mouse keratin S. for J. P. for in and S. a in the