MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism

Background

Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.

Methods and Results

siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G₀/G₁ to S. Protein expression of the cyclin-dependent kinase inhibitor p27^kip1, but not p21^cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27^kip1-/- mice indicating that the effect of MARCKS is p27^kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27^kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27^kip1 at serine 10 by KIS is required for nuclear export and degradation of p27^kip1. MARCKS knockdown caused nuclear trapping of p27^kip1. Both p27^kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.

Conclusions

MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27^kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.     

p27kip1 expression and phosphorylation dictate Palbociclib sensitivity in KRAS-mutated colorectal cancer

In colorectal cancer, mutation of KRAS (RAS^MUT) reduces therapeutic options, negatively affecting prognosis of the patients. In this setting, administration of CDK4/6-inhibitors, alone or in combination with other drugs, is being tested as promising therapeutic strategy. Identifying sensitive patients and overcoming intrinsic and acquired resistance to CDK4/6 inhibition represent still open challenges, to obtain better clinical responses. Here, we investigated the role of the CDK inhibitor p27^kip1 in the response to the selective CDK4/6-inhibitor Palbociclib, in colorectal cancer. Our results show that p27^kip1 expression inversely correlated with Palbociclib response, both in vitro and in vivo. Generating a model of Palbociclib-resistant RAS^MUT colorectal cancer cells, we observed an increased expression of p27^kip1, cyclin D, CDK4 and CDK6, coupled with an increased association between p27^kip1 and CDK4. Furthermore, Palbociclib-resistant cells showed increased Src-mediated phosphorylation of p27^kip1 on tyrosine residues and low doses of Src inhibitors re-sensitized resistant cells to Palbociclib. Since p27^kip1 showed variable expression in RAS^MUT colorectal cancer samples, our study supports the possibility that p27^kip1 could serve as biomarker to stratify patients who might benefit from CDK4/6 inhibition, alone or in combination with Src inhibitors.Subject terms: Colorectal cancer, Cell growth, Cell signalling     

A single-nucleotide polymorphism in the human p27<Superscript>kip1</Superscript>gene (-838C>A) affects basal promoter activity and the risk of myocardial infarction

Background  

Excessive proliferation of vascular smooth muscle cells and leukocytes within the artery wall is a major event in the development of atherosclerosis. The growth suppressor p27^kip1 associates with several cyclin-dependent kinase/cyclin complexes, thereby abrogating their capacity to induce progression through the cell cycle. Recent studies have implicated p27^kip1 in the control of neointimal hyperplasia. For instance, p27^kip1 ablation in apolipoprotein-E-null mice enhanced arterial cell proliferation and accelerated atherogenesis induced by dietary cholesterol. Therefore, p27^kip1 is a candidate gene to modify the risk of developing atherosclerosis and associated ischaemic events (i.e., myocardial infarction and stroke).     

Cellular thiol status-dependent inhibition of tumor cell growth via modulation of p27<Superscript>kip1</Superscript> translocation and retinoblastoma protein phosphorylation by 1′-acetoxychavicol acetate

Summary. 1′-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27^kip1, which is an important regulator of the mammalian cell cycle, we estimated the amount of p27^kip1 levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27^kip1 levels, but caused a decrease in phosphorylated p27^kip1 and an increase in unphosphorylated p27^kip1 as well as an increase in the nuclear localization of p27^kip1. These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27^kip1 phosphorylation and an increase in nuclear localization of p27^kip1, and these events are dependent on the cellular thiol status.     

The cyclin-dependent kinase inhibitor p27kip1 is required for transplantation tolerance induced by costimulatory blockade

The cyclin-dependent kinase (CDK) inhibitor p27kip1 is an important negative regulator of the cell cycle that sets a threshold for mitogenic signals in T lymphocytes, and is required for T cell anergy in vitro. To determine whether p27(kip1) is required for tolerance in vivo, we performed cardiac allograft transplantation under conditions of combined CD28/CD40L costimulatory blockade. Although this treatment induced long-term allograft survival in wild-type recipients, costimulatory blockade was no longer sufficient to induce tolerance in mice lacking p27kip1. Rejected allografts from p27kip1-/- mice contained more CD4+ T lymphocytes and exhibited more tissue damage than allografts from tolerant, wild-type mice. Infiltrating p27kip1-deficient T cells, but not wild-type T cells, exhibited nuclear expression of cyclins E and A, indicating uncontrolled T cell cycle progression in the graft. The failure of tolerance in p27kip1-/- mice was also accompanied by markedly increased numbers of allospecific, IFN-gamma-producing cells in the periphery, and occurred despite apparently normal regulatory T cell activity. These data demonstrate that the CDK inhibitor p27kip1 enforces the costimulatory requirement for the expansion and differentiation of alloimmune effector T lymphocytes in vivo, and point to CDKs as novel targets for immunosuppressive or tolerance-inducing therapies.     

Downregulation of p57kip2 promotes cell invasion via LIMK/cofilin pathway in human nasopharyngeal carcinoma cells

The members of Rho family are well known for their regulation of actin cytoskeleton to control cell migration. The Cip/kip members of cyclin‐dependent (CDK) inhibitors have shown to implicate in cell migration and cytoskeletal dynamics. p57^kip2, a CDK inhibitor, is frequently down‐regulated in several malignancy tumors. However, its biological roles in human nasopharyngeal carcinoma (NPC) cells remained to be investigated. Here, we found p57^kip2 has nuclear and cytoplasm distributions and depletion of endogenous p57^kip2 did not change the cell‐cycle progression. Inhibition of cell proliferation by mitomycin C promoted FBS‐mediated cell migration and accompanied with the downregulation of ΔNp63α and p57^kip2, but did not change the level of p27^kip1, another CDK inhibitor. By using siRNA transfection and cell migration/invasion assays, we found that knockdown of p57^kip2, but not ΔNp63α, involved in promotion of NPC cell migration and invasion via decrease of phospho‐cofilin (p‐cofilin). Treatment with Y‐27632, a specific ROCK inhibitor, we found that dysregulation of ROCK/cofilin pathway decreased p‐cofilin expression and induced cell migration. This change of p‐cofilin induced actin remodeling and pronounced increase of membrane protrusions. Further, silence of p57^kip2 not only decreased the interaction between p57^kip2 and LIMK‐1 assayed by immunoprecipitation but also reduced the level of phospho‐LIMK1/2. Therefore, this study indicated that dysregulation of p57^kip2 promoted cell migration and invasion through modulation of LIMK/cofilin signaling and suggested this induction of inappropriate cell motility might contribute to promoting tumor cell for metastasis. J. Cell. Biochem. 112: 3459–3468, 2011. © 2011 Wiley Periodicals, Inc.     

The non-canonical functions of p27Kip1 in normal and tumor biology

p27^Kip1 was first discovered as a key regulator of cell proliferation. The canonical function of p27^Kip1 is inhibition of cyclin-dependent kinase (CDK) activity. In addition to its initial identification as a CDK inhibitor, p27^Kip1 has also emerged as an intrinsically unstructured, multifunctional protein with numerous non-canonical, CDK-independent functions that exert influence on key processes such as cell cycle regulation, cytoskeletal dynamics and cellular plasticity, cell migration, and stem-cell proliferation and differentiation. Many of these non-canonical functions, depending on the cell-specific contexts such as oncogenic activation of signaling pathways, have the ability to turn pro-oncogenic in nature and even contribute to tumor-aggressiveness and metastasis. This review discusses the various non-canonical, CDK-independent mechanisms by which p27^Kip1 functions either as a tumor-suppressor or tumor-promoter.     

Dynamic Changes of p27<Superscript>kip1</Superscript> and Skp2 Expression in Injured Rat Sciatic Nerve

S phase kinase-associated protein 2 (Skp2), an F-box protein, is required for the ubiquitination and consequent degradation of p27^kip1. Previous reports have showed that p27^kip1 played important roles in cell cycle regulation and neurogenesis in the developing central nervous system. But the distribution and function of p27^kip1 and Skp2 in nervous system lesion and regeneration remains unclear. In this study, we observed that they were expressed mainly in both Schwann cells and axons in adult rat sciatic nerve. Sciatic nerve crush and transection resulted in a significant up-regulation of Skp2 and a down-regulation of p27^kip1. By immunochemistry, we found that in the distal stumps of transected nerve from the end to the edge, the appearance of Skp2 in the edge is coincided with the decrease in p27^kip1 levels. Changes of them were inversely correlated. Results obtained by coimmunoprecipitation and double labeling further showed their interaction in the regenerating process. Thus, these results indicate that p27^kip1 and Skp2 likely play an important role in peripheral nerve injury and regeneration. Ai-Guo Shen and Shu-Xian Shi contributed equally to this work.     

Cyclin D2 compensates for the loss of cyclin D1 in estrogen-induced mouse uterine epithelial cell proliferation

The cell cycle-regulatory protein, cyclin D1, is the sensor that connects the intracellular cell cycle machinery to external signals. Given this central role in the control of cell proliferation, it was surprising that mice lacking the cyclin D1 gene were viable and fertile. Fertility requires 17beta-estradiol (E2)-induced uterine luminal epithelial cell proliferation. In these cells E2 causes the translocation of cyclin D1/cyclin-dependent kinase 4 (CDK4) from the cytoplasm into the nucleus with the consequent phosphorylation of the retinoblastoma protein. In cyclin D1 null mice, E2 also induces retinoblastoma protein phosphorylation and DNA synthesis in a normal manner. CDK4 activity was slightly reduced in the D1 null mice compared with wild-type mice. This CDK4 activity was due to complexes of cyclin D2/CDK4. Cyclin D2 was translocated into the nucleus in response to E2 in the cyclin D1-/- mice to a much greater degree than in wild-type mice. This cyclin D2/CDK4 complex was also able to bind p27kip1 in cyclin D1-/- uterine luminal epithelial cells, allowing for the activation of CDK2. Our data show that in vivo cyclin D2 can completely compensate for the loss of cyclin D1 and reinforces the conclusions that cyclin Ds are the central regulatory point in the proliferative responses of epithelial cells to estrogens.     

Mitf functions as an in ovo regulator for cell differentiation and proliferation during development of the chick RPE

Mitf has been reported to play a crucial role in regulating the differentiation of pigment cells in homeothermal animals, i.e. the melanocytes and the retinal pigment epithelium (RPE). However, less is known about the functions of Mitf in the developing RPE. To elucidate such functions, we introduced wild-type and dominant-negative Mitf expression vectors into chick optic vesicles by electroporation. Over-expression of wild-type Mitf altered neural retina cells to become RPE-like and repressed the expression of neural retina markers in vivo. In contrast, dominant-negative Mitf inhibited pigmentation in the RPE. The percentage of BrdU-positive cells decreased during normal RPE development, which was followed by Mitf protein expression. The percentage of BrdU-positive cells decreased in the wild-type Mitf-transfected neural retina, but increased in the dominant-negative Mitf-transfected RPE. p27^kip1, one of the cyclin-dependent kinase inhibitors, begins to be expressed in the proximal region of the RPE at stage 16. Transfection of wild-type Mitf induced expression of p27^kip1, while transfection of dominant-negative Mitf inhibited p27^kip1 expression. We found that Mitf was associated with the endogenous p27^kip1 5′ flanking region. These results demonstrate for the first time “in vivo” that Mitf uniquely regulates both differentiation and cell proliferation in the developing RPE.     

p27Kip1, a double-edged sword in Shh-mediated medulloblastoma

Medulloblastoma, a brain tumor arising in the cerebellum, is the most common solid childhood malignancy. The current standard of care for medulloblastoma leaves survivors with life-long side effects. Gaining insight into mechanisms regulating transformation of medulloblastoma cells-of-origin may lead to development of better treatments for these tumors. Cerebellar granule neuron precursors (CGNPs) are proposed cells-of-origin for certain classes of medulloblastoma, specifically those marked by aberrant Sonic hedgehog (Shh) signaling pathway activation. CGNPs require signaling by Shh for proliferation during brain development. In mitogen-stimulated cells, nuclear localized cyclin dependent kinase (cdk) inhibitor p27 (Kip1) functions as a checkpoint control at the G1- to S-phase transition by inhibiting cdk2. Recent studies have suggested cytoplasmically localized p27^kip1 acquires oncogenic functions. Here, we show that p27^Kip1 is cytoplasmically localized in CGNPs and mouse Shh-mediated medulloblastomas. Tranasgenic mice bearing an activating mutation in the Shh pathway and lacking one or both p27^Kip1 alleles have accelerated tumor incidence compared to mice bearing both p27^Kip1 alleles. Interestingly, mice heterozygous for p27^Kip1 have decreased survival latency compared to p27^Kip1-null animals. Our data indicate that this may reflect the requirement for at least one copy of p27^Kip1 for recruiting cyclin D/cdk4/6 to promote cell cycle progression yet insufficient expression in the heterozygous or null state to inhibit cyclin E/cdk2. Finally, we find that mis-localized p27^Kip1 may play a positive role in motility in medulloblastoma cells. Together, our data indicate that the dosage of p27^Kip1 plays a role in cell cycle progression and tumor suppression in Shh-mediated medulloblastoma expansion.     

Phosphorylation of CDK2 at threonine 160 regulates meiotic pachytene and diplotene progression in mice

Telomere clustering is a widespread phenomenon among eukaryotes. However, the molecular mechanisms that regulate formation of telomere clustering in mammalian meiotic prophase I, are still largely unknown. Here, we show that CDK2, especially p39^cdk2, as a potential meiosis-specific connector interaction with SUN1 mediates formation of telomere clustering during mouse meiosis. The transition from CDK2 to p-CDK2 also regulates the progression from homologous recombination to desynapsis by interacting with MLH1. In addition, disappearance of CDK2 on the telomeres and of p-CDK2 on recombination sites, were observed in Sun1^−/− mice and in pachytene-arrested hybrid sterile mice (pwk×C57BL/6 F1), respectively. These results suggest that transition from CDK2 to p-CDK2 plays a critical role for regulating meiosis progression.     

CDK11p110 plays a critical role in the tumorigenicity of esophageal squamous cell carcinoma cells and is a potential drug target

Esophageal squamous cell carcinoma (ESCC) is a serious malignancy with limited options for targeted therapy. The exploration of novel targeted therapies for combating ESCC is urgently needed. Cyclin-dependent kinases (CDKs) play important roles in the progression of cancers; however, the function of CDK11^p110 (cyclin-dependent kinase 11^p110) in ESCC is still unknown. Here, we investigated the effects and molecular mechanisms of CDK11^p110 in the proliferation and growth of ESCC by examining the expression of CDK11^p110 in ESCC tissues and by detecting phenotypic changes in ESCC cells after CDK11^p110 knockdown or overexpression in vitro and in vivo. According to the tissue microarray analysis, compared with its expression level in normal tissues, the expression level of CDK11^p110 was significantly elevated in ESCC tissues; this result was in concordance with the data in TCGA (The Cancer Genome Atlas) datasets. In addition, RNAi-mediated CDK11^p110 silencing exerted a substantial inhibitory effect on the proliferation, clonogenicity and migration ability of ESCC cells. Further study indicated that CDK11^p110 knockdown arrested ESCC cells in the G2/M phase of the cell cycle and induced cell apoptosis. Moreover, stable shRNA-mediated CDK11^p110 knockdown inhibited tumor growth in an ESCC xenograft model, and overexpression of CDK11^p110 enhanced tumor growth. In addition, the Ki67 proliferation index was closely associated with the elevation or depletion of CDK11^p110 in vivo. In summary, this study provides evidence that CDK11^p110 play a critical role in the tumorigenicity of ESCC cells, which suggests that CDK11^p110 may be a promising therapeutic target in ESCC.

Abbreviations: CDKs: cyclin-dependent kinases; CDK11: Cyclin-dependent kinase 11; CDK11^p110: Cyclin-dependent kinase 11^p110, the larger isomer of cyclin-dependent kinase 11; ESCC: esophageal squamous cell carcinoma; FACS: fluorescence-activated cell sorting; FDA: the Food and Drug Administration; TCGA: The Cancer Genome Atlas; TMA: tissue microarray.     

Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration

p27(kip1), a cyclin-dependent kinase (CDK) inhibitor (CKI), generally suppresses CDK activity in proliferating cells. Although another role of p27 in cell migration has been recently suggested in vitro, the physiological importance of p27 in cell migration remains elusive, as p27-deficient mice have not shown any obvious migration-defect-related phenotypes. Here, we show that Cdk5, an unconventional neuronal CDK, phosphorylates and stabilizes p27 as an upstream regulator, maintaining the amount of p27 in post-mitotic neurons. In vivo RNA interference (RNAi) experiments showed that reduced amounts of p27 caused inhibition of cortical neuronal migration and decreased the amount of F-actin in the processes of migrating neurons. The Cdk5-p27 pathway activates an actin-binding protein, cofilin, which is also shown to be involved in cortical neuronal migration in vivo. Our findings shed light on a previously unknown new relationship between CDK and CKI in G0-arrested cells that regulates cytoskeletal reorganization and neuronal migration during corticogenesis.     

The Accumulation of Cyclin-Dependent Kinase Inhibitor p27Is a Primary Response to Staurosporine and Independent of G1 Cell Cycle Arrest

The staurosporine-induced G1 cell cycle arrest was analyzed in a variety of cell lines which includes human tumor cell lines and oncogene-transformed NIH3T3 cell lines. All the cell lines which were sensitive to staurosporine-induced G1 arrest contained a functional retinoblastoma protein (pRB). However, when pRB-lacking fibroblast cells derived from pRB knockout mice were tested they were also sensitive to G1 arrest by staurosporine, indicating that the inactivation of pRB alone is not sufficient for the abrogation of staurosporine-induced G1 arrest. In searching for a common event caused by staurosporine, the cyclin-dependent kinase (CDK) inhibitor protein p27^kip1but not p21^cip1was found to accumulate after staurosporine treatment in all the cell lines examined. This accumulation occurred regardless of the induction of the G1 arrest. The result indicates that the accumulation of p27^kip1is the cell's primary response to staurosporine and that the capability of staurosporine to induce G1 arrest depends on the integrity of cell cycle regulatory components which are downstream of p27^kip1.