Inhibition of protein kinase B (PKB) and PKCzeta mediates keratin K10-induced cell cycle arrest

The intermediate filament cytoskeleton is composed of keratins in all epithelial cells and imparts mechanical integrity to these cells. However, beyond this shared function, the functional significance of the carefully regulated tissue- and differentiation-specific expression of the large keratin family of cytoskeletal proteins remains unclear. We recently demonstrated that expression of keratin K10 or K16 may regulate the phosphorylation of the retinoblastoma protein (pRb), inhibiting (K10) or stimulating (K16) cell proliferation (J. M. Paramio, M. L. Casanova, C. Segrelles, S. Mittnacht, E. B. Lane, and J. L. Jorcano, Mol. Cell. Biol. 19:3086-3094, 1999). Here we show that keratin K10 function as a negative modulator of cell cycle progression involves changes in the phosphoinositide 3-kinase (PI-3K) signal transduction pathway. Physical interaction of K10 with Akt (protein kinase B [PKB]) and atypical PKCzeta causes sequestration of these kinases within the cytoskeleton and inhibits their intracellular translocation. As a consequence, the expression of K10 impairs the activation of PKB and PKCzeta. We also demonstrate that this inhibition impedes pRb phosphorylation and reduces the expression of cyclins D1 and E. Functional and biochemical data also demonstrate that the interaction between K10 and these kinases involves the non-alpha-helical amino domain of K10 (NTerm). Together, these results suggest new and essential roles for the keratins as modulators of specific signal transduction pathways.     

Cooperation between different forms of the human papillomavirus type 1 E4 protein to block cell cycle progression and cellular DNA synthesis

Posttranslational modification-oligomerization, phosphorylation, and proteolytic cleavage-of the human papillomavirus (HPV) E4 protein occurs as the infected keratinocytes migrate up through the suprabasal wart layers. It has been postulated that these events modify E4 function during the virus life cycle. In HPV type 1 (HPV1)-induced warts, N-terminal sequences are progressively cleaved from the full-length E4 protein (E1(wedge)E4) of 17 kDa to produce a series of polypeptides of 16, 11 and 10 kDa. Here, we have shown that in human keratinocytes, a truncated protein (E4-16K), equivalent to the 16-kDa species, mediated a G(2) arrest in the cell cycle that was dependent on a threonine amino acid in a proline-rich domain of the protein. Reconstitution of cyclin B1 expression in E4-16K cells reversed the G(2) arrest. Expression of E4-16K also induced chromosomal rereplication, and this was associated with aberrant nuclear morphology. Perturbation of the mitotic cell cycle was a biological activity specific to the truncated protein. However, coexpression of the full-length E1(wedge)E4 protein and the truncated E4-16K protein inhibited normal cellular proliferation and cellular DNA rereplication but did not prevent cells from arresting in G(2). Our findings provide the first evidence to support the hypothesis that proteolytic cleavage of the E1(wedge)E4 protein modifies its function. Also, different forms of the HPV1 E4 protein cooperate to negatively influence keratinocyte proliferation. We predict that these distinct biological activities of E4 act to support efficient amplification of the viral genome in suprabasal keratinocytes.     

Expression of retinoblastoma gene product in respiratory epithelium and sinonasal neoplasms: relationship with p16 and cyclin D1 expression

Transition from G1 to S phase of the cell cycle is mediated by interactions between the Retinoblastoma gene product (pRb), p16, and cyclin D1. To determine the expression of these proteins in the sinonasal mucosa immunohistochemistry was carried out on archived tissue sections from 46 patients (37 men, 9 women, age range 17 to 82 years, median 55 years). Nuclear immunostaining for these proteins was assessed and the expression rates (percentages of immunoreactive nuclei) in normal respiratory epithelium, inverted sinonasal papillomas, cylindrical (oncocytic) sinonasal papillomas, and squamous cell carcinomas were compared. Normal respiratory epithelium showed significantly higher pRb expression in surface cells compared to basal cells (p < 0.05). In contrast, abundant pRb expression in surface and basal cells was detected in columnar differentiation in sinonasal papillomas and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed significantly reduced pRb expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05, respectively). Expression of p16 was detected in all epithelial cell layers of normal respiratory epithelium, sinonasal papillomas, and adjacent mucosa. Cuboidal and squamous metaplasia in inverted papillomas showed increased p16 expression in surface cells compared to columnar epithelium in inverted papillomas (p < 0.05 between squamous metaplasia and columnar epithelium). Sinonasal squamous cell carcinomas showed the coexpression of pRb and p16. Expression rates of cyclin D1 higher than 10% were detected only in invasive carcinomas but not in carcinoma in situ, sinonasal papillomas or respiratory epithelium. Conclusively, pRb expression accompanies terminal differentiation in columnar surface cells. Expression of pRb in proliferating basal cells is present in sinonasal papillomas and adjacent mucosa but not in normal respiratory epithelium. Cuboidal and squamous metaplasia in inverted papillomas involves downregulation of pRb expression along with increased p16 expression in surface cells. Sinonasal squamous cell carcinomas coexpress pRb and p16. Overexpression of cyclin D1 in sinonasal lesions is confined to invasive squamous cell carcinomas.     

Transient synthesis of K6 and K16 keratins in regenerating rabbit corneal epithelium: keratin markers for an alternative pathway of keratinocyte differentiation

Cultured rabbit corneal epithelial cells undergo three distinct stages of growth and differentiation characterized by the sequential appearance of K5/K14 keratin markers for basal keratinocytes, K6/K16 keratin markers for "hyperproliferative" keratinocytes, and K3/K12 keratin markers for corneal-type differentiation. Analyses of [35S]methionine-labeled, newly synthesized keratins revealed that K6/K16 are synthesized only briefly when the cells undergo exponential growth, and their synthesis is suppressed when the cells reach confluence and switch to synthesizing K3/K12. Transient synthesis of K6/K16 was also observed in vivo during corneal epithelial regeneration. Although K6/K16 expression in general correlates well with cellular growth, drug-induced inhibition of corneal epithelial growth and related data on human epidermal keratinocytes indicate that these two events are dissociable. These results establish clearly for the first time a reciprocal relationship, on a protein level, between the synthesis of K6/K16 and a differentiation-related keratin pair, K3/K12. Such a relationship strongly suggests a competitive mechanism controlling the synthesis of these two major classes of keratins in the suprabasal compartment. Our results also indicate that although hyperproliferation is usually accompanied by K6/K16 expression, the reverse is not always true. Taken together, the data suggest that K6/K16 are synthesized, perhaps by default, as an alternative suprabasal keratin pair under conditions that are nonpermissive for keratinocytes to express their normal, differentiation-related keratin pairs.     

Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HACAT and in ras-transfected tumorigenic clones

The spontaneous human keratinocyte line HaCaT and c-Ha-ras oncogene-transfected cell clones are capable of expressing an unusually broad spectrum of keratins, not observed so far in epithelial cells. This expression is, however, strongly modulated by environmental conditions, including cell density. Both cells of the nontumorigenic HaCaT line and the tumorigenic HaCaT-ras clones, I-7 and II-3 (giving rise to benign and malignant tumors, respectively), constitutively expressed the keratins K5, K6, K14, K16 and K17, which are also common in cultures of normal keratinocytes. In addition keratins K7, K8, K18 and K19, generally associated with simple epithelia, were synthesized (to a most pronounced extent in sparse cultures), while keratins K4, K13 and K15 appeared at confluence, presumably with the onset of stratification. Moreover, in both HaCaT and HaCaT-ras clones the epidermal "suprabasal" keratins, K1 and K10, were expressed in conventional submerged cultures (at normal vitamin A levels), markedly rising with cell density, but not strictly correlated with the degree of stratification. This property was maintained in HaCaT cells up to the highest passages. According to immunofluorescence, this was due to increasing numbers of strongly stained cells, and not due to a gradual increase in all cells. Most strikingly, there was a significant delay in the appearance of K10 compared to K1, and this dissociation of expression was most evident in dispase-detached cell sheets (submerged cultures) and organotypic cultures of the ras clones (grown at the air-liquid interface). While on frozen sections bright staining for K1 was seen in some basal and virtually all suprabasal cell layers, K10 was largely restricted to the uppermost layers. Thus, obviously synthesis of K1 and K10 can be regulated independently, although generally in this given sequence. The apparent compatibility of K1 synthesis with proliferation and particularly the extended delay of K10 expression (as a postmitotic event) might be causally related to altered growth control and as such imply the significance of this disturbance. Finally, the highly preserved epidermal characteristics, in terms of expression of keratins (and other differentiation markers [5]) and their regulation, makes these cell lines excellent candidates for studying external modulators of differentiation and also underlying molecular mechanisms.     

Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.

Cells which are highly proliferative typically lack expression of differentiated, lineage-specific characteristics. Id2, a member of the helix-loop-helix (HLH) protein family known to inhibit cell differentiation, binds to the retinoblastoma protein (pRb) and abolishes its growth-suppressing activity. We found that Id2 but not Id1 or Id3 was able to bind in vitro not only pRb but also the related proteins p107 and p130. Also, an association between Id2 and p107 or p130 was observed in vivo in transiently transfected Saos-2 cells. In agreement with these results, expression of Id1 or Id3 did not affect the block of cell cycle progression mediated by pRb. Conversely, expression of Id2 specifically reversed the cell cycle arrest induced by each of the three members of the pRb family. Furthermore, the growth-suppressive activities of cyclin-dependent kinase inhibitors p16 and p21 were efficiently antagonized by high levels of Id2 but not by Id1 Id3. Consistent with the role of p16 as a selective inhibitor of pRb and pRb-related protein kinase activity, p16-imposed cell cycle arrest was completely abolished by Id2. Only a partial reversal of p21-induced growth suppression was observed, which correlated with the presence of a functional pRb. We also documented decreased levels of cyclin D1 protein and mRNA and the loss of cyclin D1-cdk4 complexes in cells constitutively expressing Id2. These data provide evidence for important Id2-mediated alterations in cell cycle components normally involved in the regulatory events of cell cycle progression, and they highlight a specific role for Id2 as an antagonist of multiple tumor suppressor proteins.     

Mechanisms that limit the in vitro proliferative potential of human CD8+ T lymphocytes

Human T lymphocytes can be numerically expanded in vitro only to a limited extent. The cyclin-dependent kinase inhibitor p16(INK4a) is essential in the control of cellular proliferation, and its expression, in epithelial cells, is associated with irreversible growth arrest. Using long-term cultured CD8+ T lymphocytes, we have investigated the role of the p16/pRb pathway in the regulation of T cell proliferation and senescence. In this study, we describe at least two mechanisms that cause replicative growth arrest in cultured lymphocytes. The first one depends on the expression of p16(INK4a) and is directly responsible for the exit of a significant proportion of CD8+ T cells from the proliferative population. This induced p16 expression pattern is observed during each round of mitogen stimulation and is not related to activation-induced cell death. Importantly, knocking down p16(INK4a) expression allows increased proliferation of T cells. The second one is a phenomenon that resembles human fibroblast senescence, but is independent of p16(INK4a) and of telomere attrition. Interestingly, virtually all pRb proteins in the senescent population are found in the active form. Our data indicate that newly synthesized p16(INK4a) limits the proliferation of T lymphocytes that respond to mitogen, but is not required for the loss of mitogen responsiveness called senescence.     

The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells

When cells from normal human epidermis and from the human squamous cell carcinoma line SCC-13 were seeded on floating rafts of collagen and fibroblasts, they stratified and underwent terminal differentiation. Although the program of differentiation in SCC-13 cells was morphologically abnormal, the cultures resembled normal epidermal raft cultures by expressing the terminal differentiation-specific keratins, K1/K10, and by restricting their proliferative capacity to the basal-like cells of the population. In addition, the differentiating cells of both normal and SCC-13 raft cultures expressed keratins K6 and K16, which are not normally expressed in epidermis, but are synthesized suprabasally during wound-healing and in various epidermal diseases associated with hyperproliferation. While the behavior of normal and SCC-13 rafts was quite similar when they were cultured over normal medium, significant biochemical differences began to emerge when the cultures were exposed to retinoic acid. Most notably, while the SCC-13 cultures still stratified extensively, they showed a marked inhibition of both abnormal (K6/K16) and normal (K1/K10) differentiation-associated keratins, concomitantly with an overall disappearance of differentiated phenotype. Surprisingly, the reduction in K6/K16 in retinoid-treated SCC-13 cultures was not accompanied by a decrease in cell proliferation. Using immunohistochemistry combined with [3H]thymidine labeling, we demonstrate that while the expression of K6 and K16 are often associated with hyperproliferation, these keratins are only produced in the nondividing, differentiating populations of proliferating cultures. Moreover, since their expression can be suppressed without a corresponding decrease in proliferation, the expression of these keratins cannot be essential to the nature of the hyperproliferative epidermal cell.     

Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis

The retinoblastoma gene product, pRb, plays a crucial role in cell cycle regulation, differentiation and inhibition of oncogenic transformation. pRb and its closely related family members p107 and p130 perform exclusive and overlapping functions during mouse development. The embryonic lethality of Rb-null animals restricts the phenotypic analysis of these mice to mid-gestation embryogenesis. We employed the Cre/loxP system to study the function of Rb in adult mouse stratified epithelium. Rb(F19/F19);K14cre mice displayed hyperplasia and hyperkeratosis in the epidermis with increased proliferation and aberrant expression of differentiation markers. In vitro, pRb is essential for the maintainance of the postmitotic state of terminally differentiated keratinocytes, preventing cell cycle re-entry. However, p107 compensates for the effects of Rb loss as the phenotypic abnormalities of Rb(F19/F19);K14cre keratinocytes in vivo and in vitro become more severe with the concurrent loss of p107 alleles. p107 alone appears to be dispensable for all these phenotypic changes, as the presence of a single Rb allele in a p107-null background rescues all these alterations. Luciferase reporter experiments indicate that these phenotypic alterations might be mediated by increased E2F activity. Our findings support a model in which pRb in conjunction with p107 plays a central role in regulating epidermal homeostasis.     

Cyclin E and c-Myc promote cell proliferation in the presence of p16INK4a and of hypophosphorylated retinoblastoma family proteins.

Retroviral expression of the cyclin-dependent kinase (CDK) inhibitor p16(INK4a) in rodent fibroblasts induces dephosphorylation of pRb, p107 and p130 and leads to G1 arrest. Prior expression of cyclin E allows S-phase entry and long-term proliferation in the presence of p16. Cyclin E prevents neither the dephosphorylation of pRb family proteins, nor their association with E2F proteins in response to p16. Thus, cyclin E can bypass the p16/pRb growth-inhibitory pathway downstream of pRb activation. Retroviruses expressing E2F-1, -2 or -3 also prevent p16-induced growth arrest but are ineffective against the cyclin E-CDK2 inhibitor p27(Kip1), suggesting that E2F cannot substitute for cyclin E activity. Thus, cyclin E possesses an E2F-independent function required to enter S-phase. However, cyclin E may not simply bypass E2F function in the presence of p16, since it restores expression of E2F-regulated genes such as cyclin A or CDC2. Finally, c-Myc bypasses the p16/pRb pathway with effects indistinguishable from those of cyclin E. We suggest that this effect of Myc is mediated by its action upstream of cyclin E-CDK2, and occurs via the neutralization of p27(Kip1) family proteins, rather than induction of Cdc25A. Our data imply that oncogenic activation of c-Myc, and possibly also of cyclin E, mimics loss of the p16/pRb pathway during oncogenesis.     

Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins

Monospecific antibodies to mouse epidermal keratins were generated in rabbits and guinea pigs by injecting synthetic peptides of unique keratin sequences. The sequences were deduced from nucleotide sequences of cDNA clones representing basal (K14) and suprabasal (K1 and K10) cell-specific and hyperproliferative (K6) keratins of both the type-I and type-II subclasses. By applying single-and double-label immunofluorescence analysis, the expression of keratin peptides was analyzed in cultured keratinocytes maintained in the basal or suprabasal cell phenotypes. These cell types were selected by growth in medium containing 0.05 mM Ca2+ (basal cell) or 1.4 mM Ca2+ (suprabasal cell). The cultured basal cells expressed K6 and K14, but less than 1% expressed K1 and K10. Within a few hours after being placed in 1.4 mM Ca2+, K1 expression was observed, and by 24 h, 10%-17% of the cells expressed K1. K10 expression appeared to lag behind K1 expression, with only 5%-10% of cells in 1.4 mM Ca2+ exhibiting K10 immunoreactivity. Double-labeling studies indicated that virtually all K10-positive cells also expressed K1, while only about one-half of the K1-positive cells expressed K10. The treatment of basal cells with retinoic acid at pharmacological concentrations prevented the expression of K1 and K10 when cells were challenged by 1.4 mM Ca2+. Similarly, the introduction of the v-rasH oncogene into basal cells by a defective retroviral vector prevented the expression of suprabasal keratins in 1.4 mM Ca2+ medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16

Injury to stratified epithelia causes a strong induction of keratins 6 (K6) and 16 (K16) in post-mitotic keratinocytes located at the wound edge. We show that induction of K6 and K16 occurs within 6 h after injury to human epidermis. Their subsequent accumulation in keratinocytes correlates with the profound reorganization of keratin filaments from a pan-cytoplasmic distribution to one in which filaments are aggregated in a juxtanuclear location, opposite to the direction of cell migration. This filament reorganization coincides with additional cytoarchitectural changes and the onset of re-epithelialization after 18 h post-injury. By following the assembly of K6 and K16 in vitro and in cultured cells, we find that relative to K5 and K14, a well- characterized keratin pair that is constitutively expressed in epidermis, K6 and K16 polymerize into short 10-nm filaments that accumulate near the nucleus, a property arising from K16. Forced expression of human K16 in skin keratinocytes of transgenic mice causes a retraction of keratin filaments from the cell periphery, often in a polarized fashion. These results imply that K16 may not have a primary structural function akin to epidermal keratins. Rather, they suggest that in the context of epidermal wound healing, the function of K16 could be to promote a reorganization of the cytoplasmic array of keratin filaments, an event that precedes the onset of keratinocyte migration into the wound site.     

Three distinct keratinocyte subtypes identified in human oral epithelium by their patterns of keratin expression in culture and in xenografts

We have characterized the cells that form the human oral epithelia by analyzing their patterns of keratin expression in culture and in transplants. Keratinocytes of all oral regions synthesized high levels of keratins K5/K14 and K6/K16,K17, as expressed by cells of all stratified squamous epithelia in culture. However, cells from different regions varied in their expression in culture of retinoid-inducible (K19 and K13) and simple epithelial (K7, K8 and K18) keratins. By these criteria, all oral cells could be classified as belonging to one of three intrinsically distinct subtypes: "keratinizing" (gingiva, hard palate), "typical nonkeratinizing" (inner cheek, floor of mouth, ventral tongue) and "special non-keratinizing" (soft palate), all of which differed from the epidermal keratinocyte subtype. Cells from fetal floor of mouth expressed a pattern of keratins in culture markedly different from that of adult floor of mouth cells but identical to that of the adult "special nonkeratinizing" subtype and similar to that of several oral squamous cell carcinoma lines. When cultures of oral keratinocytes were grafted to the dermis of nude mice, they formed stratified epithelial structures after 10 days. In some areas of the stratified structures, the basal layer recapitulated the K19 expression pattern of the oral region from which they had originated. Thus, regional differentiation of the oral epithelium is based on an intrinsic specialization of regional keratinocyte stem cells. Additionally, oral cell transformation either frequently involves reversion to the fetal keratin program or else oral cells that express this keratin program are especially susceptible to transformation.     

Keratinocyte migration, proliferation, and differentiation in chronic ulcers from patients with diabetes and normal wounds.

Epithelialization of normal acute wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore barrier function. The keratinocytes in the epidermis of chronic ulcers fail to execute this series of events. To better understand the epithelial dynamics of chronic ulcers, we used immunohistochemistry to evaluate proliferation, differentiation, adhesion, and migration in keratinocytes along the margin of chronic ulcers from patients with diabetes mellitus. We compared these features with keratinocytes from the migrating epithelial tongues of acute incisional and excisional wounds from normal volunteers. Keratinocytes at the chronic ulcer edge are highly proliferative (Ki67 proliferation marker), have an activated phenotype (K16), do not stain for keratins involved in epidermal differentiation (K10 and K2), and show a reduced expression of LM-3A32 (uncleaved, precursor of the alpha3 chain of laminin 5), a key molecule present on migrating epithelium. In contrast, keratinocytes in normal acute wound migrating epithelium do not express the proliferation marker Ki67 but do express K10, K2, and LM-3A32. A better understanding of molecular mechanisms involved in keratinocyte migration may lead to molecular targets for therapies for impaired wound healing.     

Cooperation between p53 and p130(Rb2) in induction of cellular senescence

To determine pathways cooperating with p53 in cellular senescence when the retinoblastoma protein (pRb)/p16INK4a pathway is defunct, we stably transfected the p16INK4a-negative C6 rat glioma cell line with a temperature-sensitive mutant p53. Activation of p53(Val-135) induces a switch in pocket protein expression from pRb and p107 to p130(Rb2) and stalls the cells in late G1, early S-phase at high levels of cyclin E. Maintenance of the arrest depends on the functions of p130(Rb2) repressing cyclin A. Inactivation of p53 in senescent cultures restores the pocket proteins to initial levels and initiates progression into S-phase, but the cells fail to resume proliferation, likely due to DNA damage becoming apparent in the arrest and activating apoptosis subsequent to the release from p53-dependent growth suppression. The data indicate that p53 can cooperate selectively with p130(Rb2) to induce cellular senescence, a pathway that may be relevant when the pRb/p16INK4a pathway is defunct.     

Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.