Published online before print June 2, 2008, doi: 10.1161/CIRCRESAHA.107.169334
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© 2008 American Heart Association, Inc.
Integrative Physiology |
Myocardial Induction of Nucleostemin in Response to Postnatal Growth and Pathological Challenge
From the San Diego State University Heart Institute and Department of Biology (S.S., N.G., J.M., M.R., G.E., J.F., M.A.S.), San Diego State University, Calif; Departments of Anesthesia and Medicine and Division of Cardiology (T.H., S.V., C.P., D.D'A., J.K., A.L., P.A.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; and Biosource International (E.S.), Hopkinton, Mass.
Correspondence to Mark Sussman SDSU Heart Institute and Department of Biology, San Diego State University, 5500 Campanile Dr, San Diego, CA 92182. E-mail sussman{at}heart.sdsu.edu
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Abstract |
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Stem cell–specific proteins and regulatory pathways that determine self-renewal and differentiation have become of fundamental importance in understanding regenerative and reparative processes in the myocardium. One such regulatory protein, named nucleostemin, has been studied in the context of stem cells and several cancer cell lines, where expression is associated with proliferation and maintenance of a primitive cellular phenotype. We find nucleostemin is present in young myocardium and is also induced following cardiomyopathic injury. Nucleostemin expression in cardiomyocytes is induced by fibroblast growth factor-2 and accumulates in response to Pim-1 kinase activity. Cardiac stem cells also express nucleostemin that is diminished in response to commitment to a differentiated phenotype. Overexpression of nucleostemin in cultured cardiac stem cells increases proliferation while preserving telomere length, providing a mechanistic basis for potential actions of nucleostemin in promotion of cell survival and proliferation as seen in other cell types.
Key Words: nucleostemin • cardioprotection • cardiomyocytes • stem cells • Pim-1 • telomerase
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Introduction |
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Cellular-based myocardial regeneration depends on tightly regulated signaling cascades that control survival and proliferation. In the case of stem cell populations, these signaling pathways have been predominantly defined by decades of study in hematopoietic1–4 and developmental contexts.5–7 The relatively recent advent of myocardial adult stem cells and their distinctive characteristics has prompted reexamination of the operational definition of "stem cells" and "stemness."8,9 The traditional view of stem cell behavior as derived from classic lineage studies may not appropriately reflect the biology of stem cells in tissues characterized by slow cellular turnover such as the myocardium. For example, activation of signaling typically associated with regulation of proliferation and survival in stem cells is also observed in combination with partial or fully committed cellular phenotypes following tissue injury.10–12 These revelations have prompted dissolution of long-standing assertions related to "stem cell–associated" signaling, now viewed as regulation of tissue repair and regeneration or, in some, cases oncogenic transformation.13–16
Nucleostemin is found at high levels in various stem cells and human cancers,17 where it has been associated with maintenance of proliferation.17–20 Expression of nucleostemin drops precipitously during differentiation21,22 and genetic deletion of nucleostemin results in embryonic lethality at approximately day 4 postcoitum with blastocysts comprised of cells that fail to enter S phase.23 Similar arrest in G0/G1 phase of cell cycle was observed in HeLa cells if nucleostemin was eliminated by RNA interference.20 Nucleostemin has been purported to mediate cellular dedifferentiation and regenerative processes in newts.24 Although the molecular basis of nucleostemin-mediated actions remains controversial, evidence supports mechanisms related to inhibition of p5317 or telomeric repeat-binding factor 1 (TRF1) that negatively regulates telomere length.25 Collectively, these characteristics point to a pivotal role for nucleostemin in maintenance of cell survival, antagonizing senescence, and promotion of regenerative potential.
Participation of nucleostemin in myocardial repair and regeneration has no precedent in the literature. Our findings establish a role for nucleostemin in response to pathological injury and demonstrate biological properties of nucleostemin expression in cardiac stem cells (CSCs), postnatal development, and response to paracrine fibroblast growth factor (FGF) treatment, as well as induction by Pim-1 kinase activity. Beneficial action depends on enhanced cell proliferation coupled with maintenance of telomeric length, which is preserved in c-kit+ CSCs by nucleostemin overexpression. Therefore, nucleostemin is a novel marker of protective signaling in the myocardium that, together with established links to stem cells, point to a role in myocardial repair and regeneration.
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Materials and Methods |
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Details regarding nucleostemin cDNA, adenovirus, FGF treatments, antibodies, and immunoblotting are provided in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
Immunohistochemistry and confocal microscopy, including cell proliferation and telomere length measurements, were performed as previously described,26,27 with details in the online data supplement.
Stem cell and adult cardiomyocyte cultures were performed as described previously,28,29 with details in the online data supplement.
Murine surgical procedures were performed as previously described,30 with details provided in the online data supplement.
Pim-1–overexpressing transgenic mice were created as previously described.31
All data are expressed as means±SEM. Differences in variables examined by Student t test. P<0.05 was considered significant. Statistical data analyzed using Microsoft Excel software.
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Results |
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Nucleostemin Expression Declines Rapidly After Birth
Nucleostemin is detectable within nuclei of cardiomyocytes in sections of neonatal mouse myocardium, as well as cultured neonatal rat cells (Figure 1). Nucleostemin expression diminishes rapidly within weeks after birth evidenced by fewer positive nuclei with lower intensity immunofluorescence in sections of older hearts relative to postnatal sections (Figure 1A). Nucleoli of cultured cardiomyocytes are labeled consistent with nucleostemin localization (Figure 1B).32 Progressive loss of nucleostemin correlated with increased age in myocardial sections (Figure 1A) and lysates showing significant (P<0.01) decreases in nucleostemin protein (Figure 1C). These results indicate nucleostemin association with young myocytes possessing proliferative potential during early postnatal growth.33–36 Exposure of neonatal rat cardiomyocytes to doxorubicin significantly decreases nucleostemin protein levels (Figure IA in the online data supplement), indicating cardiotoxic effects of doxorubicin may impair proliferation of young myocytes through antagonizing nucleostemin. However, nucleostemin overexpression is ineffective at antagonizing p53 protein in this system (supplemental Figure IB).
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Nucleostemin Is Induced Following Pathological Challenge
Relatively low-level nucleostemin expression in adult myocardium is markedly increased by acute pathological challenge or chronic heart failure (Figure 2). Myocardial infarction prompts nucleostemin expression in nuclei of cardiomyocytes primarily localized to the border zone adjacent to the ischemic region (Figure 2A). Immunoblot analyses of excised border zone/infarct regions reveal nucleostemin is increased at 24 hours after induction of myocardial infarction, with significant elevation of protein level within 48 hours that peaks at 72 hours. After 96 hours, expression of nucleostemin decreases from peak levels and returns to basal levels within 1 week (Figure 2B). In addition to cardiomyocyte expression, nucleostemin is also expressed in c-kit+ cells observed 4 days postinfarction. Two areas of enrichment for these c-kit+/nucleostemin+ cells were the endothelial layer of healthy vessels in proximity to the infarct (Figure 2C) and individual cells in proximity to the border zone of damaged tissue (Figure 2D, at arrow).
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Observations of nucleostemin expression in pathologically challenged myocardium were extended to include additional models of cardiac stress characterized by heart failure or pressure overload hypertrophy. The tropomodulin-overexpressing transgenic (TOT) mouse model is a well-characterized model of chronic dilated cardiomyopathy developed by our group.37–39 TOTs show nucleostemin expression throughout the myocardium by confocal microscopy (Figure 3A) and elevated protein level by immunoblot (Figure 3B). In comparison, pressure overload–induced hypertrophy also induced increased nucleostemin immunoreactivity in sections prepared from mice subjected to transaortic constriction. Areas of nucleostemin reactivity are restricted to cells neighboring and comprising large vessels such as endothelium lining the interior as well as cardiomyocytes surrounding vessels (Figure 3C). Quantitative immunoblot analysis of TAC-induced nucleostemin expression in the vasculature is not practical because of comparatively restricted regionalization of protein expression around large vessels relative to the whole heart.
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Nucleostemin Expression Is Induced by FGF
At present, relatively little is known about inductive signals that mediate nucleostemin expression, but FGF-2 increases nucleostemin in adult bone marrow stem cells.21 Similarly, treatment of cultured adult mouse cardiomyocytes with FGF-2 prompts induction of nucleostemin immunoreactivity (Figure 4A). Immunofluorescence microscopy of FGF-2–treated cells show relatively preserved rod-shaped morphology of the FGF-treated cultures compared to vehicle-treated cells (Figure 4A). Immunoblot analyses demonstrate significant elevation of nucleostemin protein expression that peaks within 2 hours posttreatment but returns to basal levels after 8 hours (Figure 4B and 4C). In vitro findings were validated in vivo using systemic FGF-2 delivery by osmotic pump. Myocardial sections show increased nucleostemin immunoreactivity in cardiomyocytes of mice receiving osmotic pumps with FGF-2 compared to control samples (Figure 4A). This increase in myocardial nucleostemin is significant as assessed by quantitative immunoblots (Figure 4E and 4F).
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Nucleostemin Is Expressed in Cardiac Stem Cells and Declines on Differentiation
Established association of nucleostemin with stem cells17,21 and c-kit+ cells in the myocardium (Figure 2C and 2D) prompted further assessment of nucleostemin expression in CSCs. Neonatal mouse myocardium, which is enriched for c-kit+ cells,26 shows colocalization between c-kit and nucleostemin immunoreactivity (Figure 5A). Furthermore, cultured CSCs express high levels of nucleostemin as observed by immunohistochemistry (Figure 5B), as well as immunoblot (Figure 5C). Expression of nucleostemin in CSCs is associated with maintenance of an undifferentiated phenotype. When induced to lineage commitment by exposure to dexamethasone,40 CSCs show a precipitous decline in nucleostemin expression that is statistically significant (Figure 5D), along with increased labeling for GATA-4 (Figure 5B) and loss of c-kit expression (data not shown).
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Nucleostemin Expression Is Associated With Pim-1 Kinase Activity
Recent studies from our group have identified Pim-1 kinase as an essential regulator of cell survival downstream of Akt.31 Pim-1 is associated with cell proliferation and survival in the hematopoietic system41; therefore, experiments were performed to assess the relationship between Pim-1 activity and nucleostemin expression in myocardium. Normal mice show minimal levels of Pim-1 or nucleostemin expression (Figure 6A), whereas sections from transgenic mice created to overexpress Pim-1 kinase show accumulation of nucleostemin in cardiomyocyte nuclei (Figure 6B, at arrows). Induction of nucleostemin expression is also demonstrable by immunoblot analyses of lysates created from Pim-1–overexpressing transgenics relative to nontransgenic controls (Figure 6C). Furthermore, colocalization is observable in myocardial sections from mice at 4 days after infarction challenge, where surviving myocytes in the border zone coexpress both Pim-1 and nucleostemin (Figure 6D, at arrows).
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Nucleostemin Increases TERT and Telomere Regulatory Protein Expression
The molecular basis for nucleostemin effects on telomere regulatory components was assessed by immunoblot analyses of CSC culture lysates. Nucleostemin overexpression prompted concomitant increases in levels of telomere-associated regulatory proteins TERT, TRF1, and TRF2 (supplemental Figure II). These results are consistent with preservation of telomeric length mediated by nucleostemin overexpression in cultured CSCs.
Nucleostemin Increases Cardiac Stem Cell Proliferation While Preserving Telomere Length
Effects of nucleostemin overexpression on c-kit+ cultured CSCs were studied to assess consequences for cell proliferation and preservation of telomeric length. Increased nucleostemin expression was readily detected in the CSC cultures following infection with the adenoviral vector by immunoblot (supplemental Figure III). Nucleostemin overexpression in CSC promotes increased 5-bromodeoxyuridine labeling of nuclei indicative of DNA synthesis, as well as increased cell cycle progression, as demonstrated by a greater percentage of cells labeled by Ki67 (Figure 7). The number of CSCs with telomerase detectable by immunolocalization was significantly increased following nucleostemin overexpression, corresponding with a higher percentage of proliferative cells within the telomerase positive CSC population when nucleostemin is overexpressed. Despite enhanced CSC proliferation resulting from nucleostemin overexpression, average telomeric length in the CSC population was preserved and remained unchanged relative to normal control CSCs that undergo proliferative growth at a lower rate (supplemental Figure III).
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Discussion |
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Traditional categorizations of stem cell–associated molecules, such as nucleostemin, are being redefined as these signaling cascades are discovered in partially committed or fully differentiated cells and tissues.30 Because nucleostemin is associated with cellular proliferation, it is not surprising that this pathway is activated in response to postnatal growth or pathological injury. Initially, our intent was to demonstrate expression of nucleostemin in regenerative processes associated with cardiac stem and progenitor cell populations. However, in addition to observing associations between nucleostemin and c-kit+ cells, we noted profound increases in nucleostemin activation in neonatal and pathologically challenged myocardium within cardiomyocytes, prompting additional studies to understand the role of nucleostemin signaling in response to myocardial injury and survival signaling. Since the discovery of nucleostemin 5 years ago17 subsequent literature has focused predominantly on aspects of cancer,18–20,42,43 stem cells,21,22,44,45 and developmental biology.23
Previous studies of nucleostemin establish the connection between nucleostemin and proliferative populations, either in the form of stem cells21,46 or cancer.20 In this context, nucleostemin appears to be a consistent marker for maintenance of a proliferative state, because expression is rapidly lost on commitment to a differentiated postmitotic phenotype22 and depletion of nucleostemin leads to cell cycle arrest.47 Conversely, nucleostemin expression is rapidly induced in response to regenerative growth in the newt24 and is required for embryonic development because nucleostemin-null mice die in blastocyst stage approximately 4 days after fertilization.23 Loss of nucleostemin apparently renders cells incapable of DNA synthesis completion in S phase for HeLa cell cultures.20 In the context of myocardium, nucleostemin is enriched in postnatal myocytes, as well as cultured neonatal rat cardiomyocytes, and is downregulated in adult heart (Figure 1) or in CSCs induced to differentiation (Figure 5). These findings are consistent with expression of nucleostemin in a proproliferative state as neonatal myocytes are capable of limited mitotic activity. Although nucleostemin overexpression does not stimulate proliferation or hypertrophy in cultured adult cardiomyocytes (data not shown), induction of nucleostemin expression may be useful for antagonizing telomeric shortening associated with cardiomyocyte senescence and death.48,49 In addition, increased TERT expression following nucleostemin expression in CSCs may promote cell proliferation similar to that observed for hair follicle stem cells.50 Thus, in CSCs, the presence of nucleostemin may serve important roles in maintaining proliferative potential, as well as antagonizing telomeric shortening associated with enhanced mitotic activity (Figure 7 and supplemental Figure II).
Functionally competent telomerase is restricted to a few cells in adult organism, germ cells, and stem/progenitor cells.51 In telomerase-competent cycling cells, detection of TERT in combination with markers of the cell cycle indicates that telomerase is active and prevents telomeric shortening. TERT expression is higher in nucleostemin-overexpressing CPCs than in control CPCs (Figure 7 and supplemental Figure II). Additionally, TERT and Ki67 colocalize in CPCs (Figure 7). Cycling CPCs that express TERT represent morphological counterparts of telomerase activity detectable with PCR-based methods. Importantly, the fraction of telomerase-competent cycling CPCs was higher in nucleostemin-infected CPCs, indicating that nucleostemin promotes CPC proliferation without affecting telomere length. In fact, by upregulating TERT expression, nucleostemin allows CPCs to undergo multiple divisions opposing telomere attrition. It is not surprising that length of telomeres did not differ in noninfected and nucleostemin expressing CPCs. Although nucleostemin overexpressing CPCs showed higher levels of TERT (Figure 7 and supplemental Figure II), control CPCs also possess telomerase. In physiological conditions, the function of telomerase is not to elongate telomeres beyond their physiological length but to prevent telomeric shortening. Finally, 3 to 5 days in culture is a very short time interval for the control cells that would not be expected to show detectable erosion of telomeres caused by rounds of replication.
TRF1 and TRF2 are 2 telomere-related protein components of a multiple protein complex, shelterin, that control homeostasis of telomeres by modulating access of telomerase to telomeres.52 In this regard, decreased TRF1 binding to telomeres reduce the affinity of telomerase to telomeres.53 TRF1 and TRF2 promote formation of T loops in which the telomere terminus is concealed to prevent its recognition as DNA strand break by DNA damage/repair machinery.54 This particular conformation of telomeres is nonaccessible to telomerase, thereby blocking telomere elongation. TRF1 and TRF2 are abundant in long telomeres but are absent in short telomeres, allowing telomerase to act only on short telomeres to prevent further erosion.55 Increases in TRF1 and TRF2 protein resulting from nucleostemin overexpression (supplemental Figure II) may indicate that 3' telomere termini are sequestered within the T loops opposing telomerase-dependent elongation of telomeres of normal length. TRF1 and TRF2 are critical for T-loop formation, and maintenance of this specific telomere-associated molecular structure is essential for continued cellular proliferation and prevention of senescence.
Because telomerase activity is critical for maintenance of cardiac structure and function,56 nucleostemin may act to fine-tune endogenous telomerase activity and promote maintenance of telomere length as well as inhibit p53-associated signaling resulting from shortened telomeres. These postulates would be consistent with diminution of nucleostemin following exposure of cultured cardiomyocytes to doxorubicin (supplemental Figure I), and lack of nucleostemin overexpression affecting p53 levels in this context may be explained by inhibition of MDM2 resulting from aberrant nucleostemin levels.57 Under normal circumstances where telomeric shortening is linked to senescence and possibly apoptosis,56,58,59 nucleostemin accumulation may serve to antagonize these processes in injured or aging myocardium, as implicated by increases in nucleostemin resulting from cardiomyopathic injury (Figures 2 and 3
). In the case of stem cells, nucleostemin may enable cell cycling, as would be desirable in regenerative processes resulting from tissue injury or stress as supported by association of nucleostemin with c-kit+ cells in the myocardium and cultured CSCs under proliferative conditions (Figure 5).
Signal transduction controlling nucleostemin expression is not well documented, but FGF-2 induces nucleostemin expression in bone marrow stem cells.21 Interestingly, FGF-2 exerts prosurvival effects in myocardium, is a potent angiogenic molecule, and is a crucial factor for proliferation and maintenance of several cell types, including stem cell populations.60 Interestingly, FGF-2 promotes differentiation of resident cardiac precursors into functional cardiomyocytes,61 which would seem at odds with maintenance of a proliferative state unless the action of FGF-2 occurs at an early stage of commitment, when limited mitotic activity occurs in concert with lineage specification. FGF-2 stimulates Akt activity that could account for prosurvival and proproliferative effects,62–64 and Akt activation lies upstream from Pim-1 induction in cardiomyocytes.31 Induction of nucleostemin expression by Pim-1 activity (Figure 6) is without precedent in the literature and reveals an important mechanistic basis for Pim-1–mediated promotion of proliferation in the myocardium that will require further investigation.
The expression of nucleostemin in proliferative neonatal cardiomyocytes and CSCs, together with reemergence of this protein in damaged myocardium, opens up a new facet of our understanding of reparative and regenerative signaling in the heart. Nucleostemin may be useful as a molecular interventional tool for antagonizing cellular senescence, as well as maintaining proliferation. Alternatively, nucleostemin in mature postmitotic cells such as cardiomyocytes may represent part of the reversion to a fetal or embryonic gene expression profile associated with cardiomyopathic challenge or stress. For the emerging field of CSCs, nucleostemin could be useful as a marker for identification of activated stem cells in the heart and provide a valuable marker of cellular proliferative state similar to Ki67.26,37,65 Future studies are needed to expand on these intriguing seminal observations and define relationships between nucleostemin and cell status, as well as functional effects, in myocardial cells of both multipotent and lineage-committed cell types.
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Acknowledgments |
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We thank all members of the laboratory of M.S. for helpful discussion and comments.
Sources of Funding
M.S. is supported by NIH grants 5R01HL067245, 1R01HL091102, 1P01HL085577, and 1P01AG023071 (principal investigator, P.A.). N.G. and J.M. are Fellows of the Rees-Stealy Research Foundation and the San Diego State University Heart Institute.
Disclosures
None.
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Footnotes |
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Original received December 4, 2007; revision received May 19, 2008; accepted May 21, 2008.
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