Modulation of cell proliferation by cytokeratins K10 and K16 - PubMed (original) (raw)

Modulation of cell proliferation by cytokeratins K10 and K16

J M Paramio et al. Mol Cell Biol. 1999 Apr.

Abstract

The members of the large keratin family of cytoskeletal proteins are expressed in a carefully regulated tissue- and differentiation-specific manner. Although these proteins are thought to be involved in imparting mechanical integrity to epithelial cells, the functional significance of their complex differential expression is still unclear. Here we provide new data suggesting that the expression of particular keratins may influence cell proliferation. Specifically, we demonstrate that the ectopic expression of K10 inhibits the proliferation of human keratinocytes in culture, while K16 expression appears to promote the proliferation of these cells. Other keratins, such as K13 or K14, do not significantly alter this parameter. K10-induced inhibition is reversed by the coexpression of K16 but not that of K14. These results are coherent with the observed expression pattern of these proteins in the epidermis: basal, proliferative keratinocytes express K14; when they terminally differentiate, keratinocytes switch off K14 and start K10 expression, whereas in response to hyperproliferative stimuli, K16 replaces K10. The characteristics of this process indicate that K10 and K16 act on the retinoblastoma (Rb) pathway, as (i) K10-induced inhibition is hampered by cotransfection with viral oncoproteins which interfere with pRb but not with p53; (ii) K10-mediated cell growth arrest is rescued by the coexpression of specific cyclins, cyclin-dependent kinases (CDKs), or cyclin-CDK complexes; (iii) K10-induced inhibition does not take place in Rb-deficient cells but is restored in these cells by cotransfection with pRb or p107 but not p130; (iv) K16 efficiently rescues the cell growth arrest induced by pRb in HaCaT cells but not that induced by p107 or p130; and (v) pRb phosphorylation and cyclin D1 expression are reduced in K10-transfected cells and increased in K16-transfected cells. Finally, using K10 deletion mutants, we map this inhibitory function to the nonhelical terminal domains of K10, hypervariable regions in which keratin-specific functions are thought to reside, and demonstrate that the presence of one of these domains is sufficient to promote cell growth arrest.

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Figures

FIG. 1

FIG. 1

Expression of keratin K10 inhibits cell proliferation. (A) Examples of HaCaT cells transfected with empty pcDNA3 (NEO) or the corresponding keratin-containing plasmids. (B) Summary of 5 to 10 independent experiments demonstrating that K10 inhibits cell proliferation and K16 produces an increase in the number of clones. Data are shown as means ± standard deviations. (C) Distribution of the clone sizes from transfections with pcDNA3 (Neo), K10, and K16 demonstrates that K16 clones are larger than vector clones and that these are larger than K10 clones.

FIG. 2

FIG. 2

Examples of double immunofluorescence analysis of clones isolated after K10 (A, A′, and A") and K16 (B, B′, and B") transfection. Note that K10 (A) expression is restricted to a few cells, whereas K16 (B) is expressed in most cells. Cells were stained with K8.60 antibody (A) or LL025 antibody (B). A′ and B′ are the same fields as A and B, respectively, stained with the Troma 1 anti-K8 antibody. A" and B" are the double exposures from A and A′ and B and B′, respectively, to better visualize cells positive (yellow-orange) and negative (green) for the transfected keratin.

FIG. 3

FIG. 3

K10 represses cell cycle progression in a dose-dependent manner. PtK2 simple epithelial cells were transfected with the different keratin-coding plasmids. (A to D) Immunofluorescence analysis showing that in all cases the transfected proteins were similarly expressed and integrated into the endogenous cytoskeleton, as determined by staining with k8.60 against K10 (A), AE8 against K13 (B), RCK107 against K14 (C), or LL025 against K16 (D). (E) Cell cycle phase distribution of transfected cells and nontransfected cells (control, immunofluorescence-negative cells sorted from the same experiments) analyzed in parallel by FACS after being stained with the above-mentioned antibodies and propidium iodide. (F) PtK2 cells were cotransfected with equimolar amounts of pMTHK10, in which the K10 gene is under the control of the methallothionein promoter, and CMVβ-Gal. Twenty-four hours after transfection, cells were split and cultured in parallel, and 24 h later ZnCl2 was added at the indicated concentrations for 18 h. Protein extracts were obtained and analyzed in Western blots with antibodies against K10, β-Gal, and the endogenous keratins K8 and K18. (G) In the above transfections, cells cultured on glass coverslips were incubated in the presence of 10 μM BrdU for 8 h after induction. At this time, the base analog incorporation in the transfected (β-Gal-positive) and nontransfected cells was analyzed by double immunofluorescence with antibodies against BrdU and β-Gal, and the relative inhibition was determined for each ZnCl2 concentration. Note that there is significant inhibition of BrdU incorporation at ZnCl2 concentrations of 25 μM or higher. Data in panels E and G are from triplicate experiments and are shown as means ± standard deviations.

FIG. 4

FIG. 4

K16 facilitates keratinocyte proliferation and specifically reverts the K10-induced cell growth arrest. (A) After transfection with pcDNA3 or K16, cells were cultured under selection with the indicated amounts of serum. Note the higher proliferative potential of K16-transfected cells in all the situations compared to the cells transfected with empty vector. (B) HaCaT cells were transfected with a fixed amount of K10 (Hyg) and increasing amounts of either K14 or K16 (in pcDNA3 [Neo], conferring neomycin resistance). After double selection colonies were fixed, stained, and scored. Note that K16, in contrast to K14, efficiently reverses the K10-induced inhibition. Data are from three independent experiments and are shown as means ± standard deviations.

FIG. 5

FIG. 5

Keratin-induced modulation of cell proliferation requires a functional Rb protein. (A) Coexpression of a wild-type SV40 large T Ag (pZIPTAg) but not a mutant form lacking the ability of pRb binding (pZIPk1TAg) results in recovery from the K10-induced arrest. (B) The specific coexpression of certain cyclins and/or cdk’s with K10 reverts the induced inhibition in HaCaT cells, as demonstrated by the increased number of colonies with respect to empty vector (Hyg) plus each cyclin construct. (C) K10 is unable to cause cell growth inhibition in pRb-deficient C33A cells. (D) The coexpression of pRb or p107, but not that of p130, along with K10 in C33A cells restores the ability of this keratin to block cell proliferation. (E) Coexpression of K16 overrides the growth inhibition promoted by pRb, but not that promoted by p107 or p130, in HaCaT cells. Data in panels A to E are from at least three independent experiments and are shown as means ± standard deviations. (F) Immunoblotting of protein extracts from C33A cells transiently cotransfected with the indicated plasmids, demonstrating the expression of transfected β-Gal, pRb, K10, K16, and the endogenous K19 keratins (with AE1 antibody), cyclin D1, and p16_ink4a_. Note that K10 reduces, while K16 increases, the phosphorylation of the cotransfected pRb as well as the endogenous cyclin D1 expression level.

FIG. 6

FIG. 6

The expression of SV40 T Ag (A) and K10 (A′) in a HaCaT cell line isolated from a clone obtained in cotransfection experiments (Fig. 5A). Note that the majority of these cells express K10, in contrast with the few positive cells observed in cell lines isolated from clones arising in single K10 transfections (Fig. 2A).

FIG. 7

FIG. 7

Removal of both amino and carboxyl termini abolishes the inhibitory function of K10. (A) Map of the K10 gene showing some restriction sites and the deletions generated. (B) Examples of transient transfections with the mutant proteins demonstrating that all of them except ΔCΔcoil integrate into the endogenous keratin cytoskeleton in PtK2 cells, including K10 (A), Δ3′UTR (B), ΔCΔcoil (C), ΔN (D), ΔC (E), and ΔNΔC (F). (C) Summary of five independent permanent transfection experiments in HaCaT cells with the different deletion proteins, demonstrating that only the simultaneous elimination of both the amino- and carboxyl-terminal domains of K10 abolishes K10’s ability to repress cell growth.

FIG. 7

FIG. 7

Removal of both amino and carboxyl termini abolishes the inhibitory function of K10. (A) Map of the K10 gene showing some restriction sites and the deletions generated. (B) Examples of transient transfections with the mutant proteins demonstrating that all of them except ΔCΔcoil integrate into the endogenous keratin cytoskeleton in PtK2 cells, including K10 (A), Δ3′UTR (B), ΔCΔcoil (C), ΔN (D), ΔC (E), and ΔNΔC (F). (C) Summary of five independent permanent transfection experiments in HaCaT cells with the different deletion proteins, demonstrating that only the simultaneous elimination of both the amino- and carboxyl-terminal domains of K10 abolishes K10’s ability to repress cell growth.

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