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Division of Endocrinology, The University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
Correspondence: Address all correspondence and requests for reprints to: Andrew F. Stewart, M.D., Chief, Division of Endocrinology and Metabolism, BST E-1140, The University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, Pennsylvania 15213. E-mail: stewart{at}dom.pitt.edu
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Abstract |
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 Top Abstract I. IntroductionII. Basic Cell Cycle...III. Developing Models of...IV. Are We Looking...V. Will Cell Cycle...VI. The Signaling-Cell Cycle...VII. Identifying Therapeutic...VIII. SummaryReferences |
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Because ß-cells have been viewed as being unable to proliferate, the science of ß-cell replication is undeveloped. In the past several years, however, this has begun to change at a rapid pace, and many laboratories are now focused on elucidating the molecular details of the control of cell cycle in the ß-cell. In this review, we review the molecular details of cell cycle control as they relate to the pancreatic ß-cell. Our hope is that this review can serve as a common basis and also a roadmap for those interested in developing novel strategies for enhancing ß-cell replication and improving insulin production in animal models as well as in human pancreatic ß-cells.
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I. Introduction |
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TopAbstractI. IntroductionII. Basic Cell Cycle...III. Developing Models of...IV. Are We Looking...V. Will Cell Cycle...VI. The Signaling-Cell Cycle...VII. Identifying Therapeutic...VIII. SummaryReferences |
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Although Swenne (1) reported more than 20 yr ago that ß-cells could replicate, as evidenced by tritiated thymidine incorporation and autoradiography, until recently, many or most authors felt that the mature, differentiated ß-cell was unable to replicate, or did so only very rarely. Accordingly, with a few exceptions, diabetes investigators have shied away from studying this phenomenon. Understanding the molecular control of ß-cell replication therefore remains years or decades behind the corresponding body of knowledge in the fibroblast, the keratinocyte, the intestinal epithelial cell, the lymphocyte, the cancer cell, and even the yeast cell. Indeed, as a telling example, most of the genetic models reviewed below in which cell cycle molecules have been overexpressed or "knocked out", and which have serendipitously led to ß-cell phenotypes, have been reported not by diabetologists, but by molecular oncologists or developmental biologists studying general cell cycle control in cancer or development. An early example is the observation by Hanahan and colleagues (2) (a molecular oncology group) that overexpression of T-antigen (TAg) in the ß-cells of transgenic mice leads to ß-cell hyperplasia and tumors. A more recent example is the observation by the Barbacid group (3) (also molecular oncologists) that whole body deletion in mice of a ubiquitously expressed cell cycle kinase, cyclin-dependent kinase (cdk-4), leads to a surprisingly ß-cell restricted phenotype.
One of the reasons for the widely held assumption that ß-cells do not replicate is that they normally replicate so slowly that the rate is difficult to accurately measure. Finegood et al. (4) have estimated that mouse and rat ß-cells replicate at a rate such that 3% of ß-cells replicate every 24 h in the adult. Kushner and associates (5) have suggested that the rate of ß-cell replication in adult mice is much slower, i.e., 0.07%/d. Human data are limited, but Butler et al. (6) have reported that 0.04% of human ß-cells stain for the cell cycle progression marker, Ki-67, and Kassem et al. (7) have reported, using human autopsy specimens, that far less than 1% of human ß-cells costain for insulin and Ki-67. These low rates of baseline replication can be stimulated in vitro and in vivo. For example, it has been known for many years that glucose can stimulate islet cell proliferation in vitro, and partial or subtotal pancreatectomy leads to transient diabetes, which is ultimately corrected in rodents by a compensatory increase in ß-cell mass, in part accounted for by ß-cell replication (1, 4, 8, 9, 10, 11). Also, gestation and the neonatal periods are associated with increased rate of ß-cell proliferation. For example, Kassem et al. (7) have reported that up to 4–6% costain with Ki-67 in the late gestational period, and 2% costain in the perinatal period. More recently, Dor et al. (12), using elegant cell lineage tracing methods in mice, have determined that although replication may be slow, it plays a central role in maintaining adult ß-cell mass, and in replenishing ß-cell mass after partial pancreatectomy.
Contemporaneously with the observations that ß-cells can and do replicate, albeit very slowly, a number of groups have recently begun to focus on the cellular mechanisms through which ß-cell replication is controlled. It is the goal of this review to summarize where the field of ß-cell replication stands in 2006 and to provide a perspective on the types of additional studies, questions, and challenges that remain. One important message from all of this is that the field of ß-cell replication and its control lags decades behind other cell types. Another important message is that we have yet to perform some of the most basic kinds of experiments on ß-cell cycle control.
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II. Basic Cell Cycle Machinery in the ß-Cell |
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TopAbstractI. IntroductionII. Basic Cell Cycle...III. Developing Models of...IV. Are We Looking...V. Will Cell Cycle...VI. The Signaling-Cell Cycle...VII. Identifying Therapeutic...VIII. SummaryReferences |
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, and a detailed summary of the status of each of these proteins in mouse, rat, and human islets is provided in Table 1. Key cell cycle checkpoints include the G1/S transition, and the G2/M transition. Although it remains formally possible that growth factors and other replication agonists might influence ß-cell replication at the G2/M checkpoint, a recent series of publications, together with an older literature on TAg, make it inescapably clear that the pathway controlling the G1/S checkpoint in the cell cycle, governed by retinoblastoma protein (Rb), is likely to be central to the control of ß-cell proliferation. Excellent recent reviews are available (13, 14).
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) (15, 16). In general, E2F1, -2, and -3 are regarded as activating a large number and broad spectrum of genes that lead to cell cycle progression. Conversely, E2F4, -5, and -6 are felt to be repressors of other genes that suppress cell cycle progression. E2F7 does not appear to have transcriptional activity. These proteins interact with, and are regulated by, the pocket protein family members described in the following section. In the murine islet, we have shown that E2F1 and -4 are present in the highest abundance, and E2F2, -5, -6, and -7 can be detected at the mRNA level (17). Surprisingly, despite the presumed generalized expression and fundamental functional nature of these proteins, the disruption of at least two of these presumably generalized cell cycle regulatory genes results in a very restricted and pancreatic-specific phenotype; total body disruption of E2F1 has been shown to result in loss of ß-cell function (18), and combined deletion of E2F1 together with E2F2 results in failure of exocrine and endocrine pancreas development and function (19). Clearly, we have much to learn regarding cell type-specific control of cell cycle progression.
B. pRb and the "pocket proteins"
The "pocket proteins" include pRb, p107 and p130 (Figs. 1–4). pRb, the protein that is defective or deficient in human retinoblastoma, is the most ubiquitously expressed and extensively studied member of the family. Detailed recent reviews are available (20, 21, 22). As is described in more detail below, pRb is believed to play a central role as the molecular "brakes" on cell cycle progression, enforcing G1/S arrest. Mutations in pRb or in the pathways that regulate pRb activity are by far the most common causes of cancer in humans. Pocket proteins are nuclear proteins and, as shown in Fig. 2, have a nuclear localization signal (NLS). They also contain two regions termed "A" and "B" that form a "small pocket" and a carboxy-terminal region, a "C-region," that, together with the A and B regions, forms a so-called "large pocket."
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In addition to E2Fs and HDACs, several viral cell cycle activating proteins, such as TAg, E1A, and E7, also bind to this small pocket (Fig. 3). They compete here for binding with E2Fs, displacing or liberating them from their interactions with pRb, an event that accounts in an important way for induction of cell cycle progression by these viral proteins. The C-region binds a number of additional proteins, including the cdk family. Cdks, in concert with the A, D, and E cyclins, phosphorylate pRb, and do so by interacting with this region.
The function of the N-terminal region (Fig. 3) is not clearly understood, but it appears not to be essential, for N-terminally truncated mutants of pRb appear to be able to fully replace the function of wild-type pRb.
Finally, pRb contains at least 16 different serines and threonines (Fig. 3) that are phosphorylation targets of many kinases, including, for example, cdk-2, -4, and -6, as well as phosphatidylinositol 3 (PI3) kinase (20, 21, 22). pRb phosphorylation is critical to its function, with most phosphorylations serving, for example, to inactivate pRb, disrupt the small pocket, and interfere with E2F binding, relieving E2F-responsive promoters from inhibition. One additional function of pRb appears to be to enforce or sustain genomic stability, as evidenced by the fact that pRb–/– mice have an increased rate of DNA mutation (23).
As noted above, pRb is widely believed to play a central role as the gatekeeper of, or "brakes" on, the G1/S transition checkpoint. In functional terms, disruption of the pRb gene causes early embryonic lethality, although part of this early lethality appears ascribable to placental defects (24). Homozygous loss of pRb causes embryonic death before the development of the islet, so the functional importance of pRb in the islet is not known. Surprisingly, cell cycle control is largely retained in murine embryonic fibroblasts (MEFs) lacking both pRb alleles, and this has been ascribed to complementing or redundant function of p107 (see below) (25, 26). Heterozygous loss of pRb has not been reported to be associated with islet abnormalities (27, 28).
Despite its central role in cell cycle control, until 2004, pRb had never been reported to be present in the murine, rat, or human pancreatic islet. Figure 4 demonstrates that pRb is present in human islets and can be phosphorylated by transduction of human islets with adenoviruses encoding the pRb kinase, cdk-4, in combination with cyclin D1 (see Section II.E and II.F) (29). We have reported similar studies recently using rat and murine islets (17, 29). Surprisingly, despite the obvious assumption that pRb would be essential for maintaining cell cycle arrest in the islet as elsewhere, we have recently generated mice conditionally disrupted for pRb in the ß-cell (30) and have found that there is no particularly striking phenotype. Clearly, we have much to learn regarding cell cycle control in the ß-cell.
The two other pocket proteins are p107 and p130 (for detailed reviews, see Refs. 25, 26 and 31, 32, 33, 34, 35). p107 and p130 are shown schematically in Fig. 2, in comparison to pRb. As can be seen in the figure, they are homologous with pRb and share the same A-B small pocket structure and the same C-peptide-containing large pocket structure. Much less is known about the function of p107 and p130, but it is assumed, because they share the structural features of pRb and can be phosphorylation targets of cdks as occurs for pRb, that they are functional homologs of pRb. On the other hand, they are not ubiquitously expressed; their levels do not exactly parallel those of pRb during cell cycle progression; they preferentially bind to different E2F family members from pRb; and global knockouts of both p107 and p130, at least on a C57B6 background, have very little in the way of a phenotype. Although the p107-null animals have a growth plate defect, both p107 and p130 knockout animals live, reproduce, and are generally healthy (31, 32, 33, 34, 35). In contrast, combined p107 plus p130 knockout mice have been prepared, and this results in embryonic or perinatal lethality (31, 32, 33, 34, 35). Triple knockout (pRb-, p107-, p130-null) mice also have been prepared, and these, like the pRb-null mice, are early embryonic lethal (25).
Cell cycle studies have shown that pRb–/– mouse embryonic fibroblasts (MEFs) replicate relatively normally, as do p107–/– and p130–/– MEFs, and even p107–/– plus p130–/– MEFs (25). Interestingly, even pRb–/– plus p107–/– plus p130–/– MEFs replicate relatively normally, as assessed using flow cytometry, but they fail to arrest in response to starvation, confluence, or DNA damage induced by 
-irradiation or doxorubricin (25). Thus, pocket protein family members are required for G1/S arrest in MEFs.
Recently, Sage et al. (26) have shown that whereas MEFs deficient for pRb arrest normally under conditions of starvation or senescence, they express high levels of p107 (but not p130). That is, despite widely held assumptions to the contrary, pRb is actually nonessential for maintaining cell cycle arrest during starvation or senescence, at least in embryonic fibroblasts. Speculating that the increase in p107 could presumably compensate for the retention of normal proliferation rates in pRb–/– MEFs, Sage et al. "acutely" removed pRb from pRbflox/flox MEFs, using an adeno-Cre expression virus. As predicted, this was associated with an acute and marked acceleration of cell cycle progression or loss of cell cycle arrest to starvation, as assessed using flow cytometry (26). Interestingly, this was associated with the normal low levels of p107. Over the course of a few days, however, p107 spontaneously increased, replication slowed, and cell cycle control was regained. Subsequent small interfering RNA knockdown of p107 released cells from G1/0 arrest. These studies make it clear that at least in MEFs, p107 can complement for pRb. Whether this kind of pocket protein complementation can occur in the ß-cell is unknown.
Not surprisingly, given the foregoing, there is little information in the literature regarding the presence of p107 or p130 in the ß-cell. We have recently demonstrated that both p107 and p130 are present in the murine islet (17), and p107 appears to increase in response to pRb loss in the islet (30). However, ß-cell mass and function have not been reported in p107- or p130-null mice. Their function and importance in the ß-cell thus remains unknown.
C. p53 and MDM2
The apparent specificity of the pRb pathway for G1/S control in ß-cells begins to be highlighted by combined p53–/– plus pRb–/– mice. Before this can be fully understood, a brief treatise on p53 is required. p53 is widely regarded as a tumor suppressor protein, and it is far upstream of pRb, as can be seen in Fig. 1. Like pRb, it is one of the most intensely studied proteins in the cell cycle, and this brief summary of its functions and regulation does not do justice to its extraordinarily complex cell biology. Interested reviewers are referred to recent excellent comprehensive reviews (36, 37, 38). Briefly, p53 is activated by DNA damage through mechanisms that remain incompletely understood, and it has tumor-suppressive and cell death-inducing functions that serve to either arrest or kill cells that contain DNA damage or mutations that might lead to unrestrained cell cycle progression. p53 is a transcription factor that transactivates other cell cycle proteins, such as p21 (see Section II.I). It also has direct interactions with cell death molecules in the Bcl-2/BAX/BAD pathway as well as others. p53 is both a nuclear and cytosolic protein and shuttles between both compartments. p53 levels are regulated to an important degree by an E3 ubiquitin ligase named MDM2 (Fig. 1); when MDM2 levels rise, p53 is ubiquitinated and targeted for proteasomal degradation. We and others have shown that p53 is present in the murine islet (17). Interestingly, hepatocyte growth factor (HGF) but not placental lactogen (PL) overexpression seems to activate p53 in the islet (17). We have shown that MDM2 is also present in the murine islet (17), but again, no information is available on MDM2 in ß-cell cycle control.
We can now return to the combined relevance of pRb and p53 to ß-cell cycle control. Two groups have shown that mice homozygously null for p53 develop a broad spectrum of tumors, but none of these are ß-cell tumors (insulinomas) (27, 28). Similarly, mice heterozygously null for pRb (homozygously null pRb mice cannot be studied because embryos are early embryonic lethal) also develop neoplasms, but again, none of these are ß-cell tumors (27, 28). In contrast, two laboratories have shown that crosses of p53-null mice with pRb heterozygous mice result in the frequent (up to 23% of the time) development of insulinomas (27, 28). Both authors noted that the endocrine tumors (islet plus pituitary) observed with combined pRb and p53 loss was remarkably similar to the findings in the multiple endocrine neoplasia (MEN) type 1 syndrome (see Section II.J). Insulinomas do not occur at birth but take weeks to months to develop. It is believed, therefore, that development of insulinomas ultimately requires that the second allele of pRb be lost in the islets of these mice, fulfilling Knudson’s "two-hit" hypothesis (39). Indeed, the development of insulinoma was associated with loss of the second (wild-type) pRb allele (28). These findings make it very clear that p53 and Rb are critical regulators of ß-cell cycle and that their combined absence has a surprisingly tissue-specific effect on ß-cells. Why this should apply to ß-cells in particular is completely obscure.
D. Large T-antigen
Simian virus large TAg is not a normal cell cycle protein, but can be seen to be highly relevant to ß-cell replication, now that the E2F, pRb, and p53 families have been described. Indeed, the very first ß-cell-targeted transgenic mouse employed the rat insulin II promoter (RIP) to target large TAg to ß-cells (2, 40, 41). As noted above, TAg interferes with the ability of pRb to interact with E2Fs and releases them from repression. It interacts with p53 as well. Not surprisingly, then, because TAg was well known to induce hyperplasia and tumors in many tissues, RIP-TAg mice developed islet hyperplasia followed by frank ß-cell malignancies (2, 40, 41). Over the years, Hanahan and Efrat (2, 40, 41) have prepared a variety of rodent cell lines, such as the ßTC3 cell line, based on the constitutive or regulatable delivery of TAg to ß-cells. In the current context, these cell lines and murine tumors reveal that freeing ß-cells from Rb- and p53-mediated repression leads to ß-cell proliferation and ultimately tumors, and therefore indicate that this is a key cell cycle check point in ß-cells, as in so many other cell types. One final point is that activation of the cell cycle by TAg in ß-cells need not necessarily lead to tumors of the ß-cell. Efrat (40, 41) has shown, using regulatable Tet promoters, that transient activation of TAg leads to ß-cell hyperplasia without tumor formation. Recently, Narushima et al. (42) have shown that TAg expression in human ß-cells in combination with telomerase can lead to regulatable increases in replication. In contrast, sustained activation of TAg leads to accumulated genetic and chromosomal damage events that then lead to insulinoma, at least in rodent islets (2, 40, 41). Although these studies would seem to suggest important roles for pRb and p53, it is important to bear in mind that TAg has additional cellular targets beyond pRb, so these events may be associations, but not necessarily cause and effect.
E. cdk-4 and cdk-6
Further evidence for a critical, cell-type specific role of the pRb pathway in ß-cell replication is provided by the work of Rane et al. (3) and Tsutsui et al. (43). Cdk-4 and cdk-6 are kinases that partner with the D-type cyclins (see Fig. 1 and Section II.F) and phosphorylate pRb and the other pocket proteins, leading to their inactivation and thereby cell cycle progression. As molecular oncologists interested in overall cell replication, these investigators created mice that lack cdk-4. They expected to find a generalized reduction in cell proliferation rates. To their surprise, they found that global cdk-4 deletion yielded a very restricted phenotype; cdk-4 knockout mice displayed abnormalities in only three tissues—the ovary, the testis, and the ß-cell. Islets displayed ß-cell hypoplasia, which caused diabetes and ketoacidosis (Fig. 5). These findings would appear to indicate marked specificity for the actions of the cdk-4/cyclin D pathway in the control of the cell cycle in ß-cells, but they fail to explain why or how it is so tissue-specific. More recently, we have shown that murine islets constitutively lack what one might guess is the only other cell cycle protein that might complement for cdk-4, namely cdk-6 (17). Others have shown that cdk-6 mRNA is also apparently absent in islets (44). Why murine islets should specifically lack cdk-6 is uncertain.
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We have shown that viral overexpression of cdk-4 and cyclin D1 in the mouse, rat, and human islet results in markedly increased pRb phosphorylation (Fig. 4), and also in a consequent increase in ß-cell replication, reaching almost 10-fold in human ß-cells (Fig. 6) (17, 29).
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F. The D-cyclins
There are three D-type cyclins: D1, D2, and D3 (for a recent review, see Refs. 13 and 14). The D-cyclins partner with cdk-4 or cdk-6, activating the kinase function of these cdks, leading to phosphorylation and inactivation of pRb and the other pocket proteins. Thus, acting in concert with cdk-4 or cdk-6, the D-cyclins stimulate cell cycle progression. D-cyclins can be targets of signaling molecules that activate cell cycle progression. For example, Friedrichsen et al. (47) have shown that prolactin (PRL) signaling via JAK-2-STAT5 increases cyclin D2. Activating mutations or rearrangements in the D-cyclins are common in human tumors. For example, genetic rearrangements that place cyclin D1 under the control of the PTH promoter are common causes of parathyroid adenomas (48).
In terms of islet expression, all authors would appear to agree that D1 and D2 are present in murine islets, whereas D3 has been variably reported to be present or absent, perhaps reflecting differing strains or genders of mice employed (Table 1). For example, Martin et al. (44) have reported that cyclin D3 levels are essentially undetectable in the murine islet. On the other hand, we (17) and Bernal-Mizrachi and colleagues (46) have observed that D3 is present in murine islets at both the mRNA and protein levels and appears comparable in abundance with D1 and D2. Regarding human islets, Chung et al. (49) have described a series of 64 human pancreatic endocrine tumors in which they sought abnormalities in the p53-Rb/cdk-4/cyclin D pathway. Interestingly, 43% of the 64 tumors demonstrated up-regulation of cyclin D1 at the protein, mRNA, and histochemical levels, and this applied to the insulinoma subset as well; 8 of 22 insulinomas also displayed up-regulation of cyclin D1. The cellular mechanisms responsible for the up-regulation did not appear to include structural alterations in the cyclin D1 gene, but rather likely resulted from abnormal transcriptional or posttranscriptional regulation of cyclin D1 in islet tumors. This degree of association of a single oncogene with a single tumor type is unusual outside the setting of heritable tumors and again points a very specific finger at the central importance of the pRb pathway in the control of the cell cycle in ß-cells. More recently, Chung and collaborators (50) have reported that ß-cell-targeted overexpression of cyclin D1 in transgenic mice results in ß-cell proliferation with obvious ß-cell hyperplasia. Interestingly, and in contrast to the constitutively active cdk-4 mice in the preceding section, the RIP-cyclin D1 mice do not develop islet tumors.
Georgia and Bhushan (51) and Kushner et al. (52) have also recently reported that cyclin D2 deletion markedly impairs attainment of ß-cell mass and function (Fig. 7), suggesting that at least in murine islets, D2 is physiologically the most important of the three D-cyclins. D1 loss in conjunction with D2 loss exacerbates the failure of ß-cell mass. These authors also report, like Martin et al. (44), that cyclin D3 is not present in the murine islet. Thus, of the three D-cyclins, cyclins D1 and D2 have so far been shown capable of regulating ß-cell mass, and one might infer that D3 plays little or no functional role in the murine ß-cell.
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) (29). Importantly, despite the activation of cell cycle progression, there was no reduction in ß-cell differentiation as measured using glucose-stimulated insulin secretion or expression of a panel of ß-cell markers (insulin, glucokinase, Glut-2, etc.). These observations suggest that direct or indirect induction of cyclin D1 may be useful therapeutically in augmenting islet mass and function.
G. cdk-2 and cyclins A and E
As noted above, pRb can undergo up to 16 different phosphorylations, some caused by cdk-4/-6 interactions and others by cdk-2 (Fig. 3). Thus, cdk-2, like cdk-4/-6 is a cell cycle progression kinase and agonist. Just as cdk-4/-6 partner with D-type cyclins, cdk-2 partners with, and is activated by, either cyclin A or cyclin E (Fig. 1). We have shown that cdk-2 and cyclins A and E are present in murine islets (17). However, in contrast to cdk-4 and the D-cyclins, there is no evidence that cdk-2 or cyclin E or A are particularly or disproportionately important in regulating cell cycle progression in ß-cells compared with other cells. On the other hand, it is clear that suppression of cdk-2 activity by upstream kinase inhibitors described in the following section does result in inhibition of cell cycle progression in ß-cells.
H. The INK family
Upstream of cdk-2/cyclin E and cdk-4/-6/cyclin D complexes are two families of inhibitory kinases (INKs) and cyclin inhibitory proteins (CIPs) or kinase inhibitory proteins (KIPs) (Fig. 1) (13, 14). The INKs include p15ink4b, 16ink4a, p18ink4c, and p19ink4d (superscripts are deleted from this point forward). In nonislet tissues, these proteins bind to and inhibit the activity of cdk-4/-6-cyclin D complexes and cdk-2-cyclin A/E complexes, thereby causing cell cycle inhibition or arrest. Several are well-documented tumor suppressor genes, and their loss is associated with a variety of human cancers.
Until recently, the role of the INK family in the ß-cell had been understudied. This is changing rapidly, and it is now very clear that these proteins play a large and important role in maintaining G1/0 arrest in the ß-cell. Murine islets contain all four INK family members (17) (Table 1). In functional terms, Moritani et al. have reported that overexpression of TGF-ß in the islet leads to p15 overexpression associated with islet hypoplasia and diabetes in both NOD and B6 mice (53). p16 and p19 have been deleted individually or in combination, but no adverse islet outcome was described (54). Halvorsen et al. (55) have reported that p16 is up-regulated in human islet cultures and may participate in the growth arrest that limits the expansion of human islet cultures. Interestingly, the constitutively active cdk-4 employed by Rane et al. (3) was rendered constitutively active by mutating cdk-4 amino acids that are required for p16 interaction. Franklin et al. (56) reported in 2000 that deletion of p18 has no particular islet consequences. Interestingly, however, when p18–/– mice were crossed with p27–/– mice, marked islet hyperplasia resulted (56). These observations are discussed in greater detail in the following section and in Section II.J. Clearly, p18 acting in concert with other cell cycle inhibitors is essential for maintaining cell cycle arrest in ß-cells.
I. The CIP/KIP/WAF family
This family includes p21cip, p27kip1, and p57kip2 (superscripts are deleted from this point forward). In nonislet tissues, the CIP/KIP/WAFs are generally viewed as inhibitors of cell cycle progression. They are widely expressed and inhibit cell cycle progression by binding to cdk-2, -4, and -6 complexes and preventing them from performing their kinase roles (13, 14). On the other hand, they can also serve to augment or facilitate cell cycle progression in some settings (57, 58). More specifically, the cdks, cyclins, and p21 and p27 are all synthesized in the cytoplasm, where p27 and p21 act as molecular chaperones, required for cdk-cyclin complex assembly. Moreover, whereas the cyclins and cdks lack NLSs and therefore are unable to gain nuclear access on their own, p21 and p27 each contain an NLS and employ these to transport assembled cdk-cyclin complexes into the nucleus, where they can drive cell cycle progression. Thus, the effects of the CIP/KIP family in cell cycle progression are bifunctional.
p21 is present in the murine and human ß-cell (17, 46) (Table 1). It has been shown to be up-regulated in response to ß-cell injury (59) and to be lost in human insulinomas (60). Recently, we have demonstrated that p21 is markedly and rapidly increased, in response to cell cycle activation in murine and human ß-cells by growth factors such as HGF, PL, PRL, and PTH-related protein (PTHrP), and translocates from the cytosol to the nucleus of human ß-cells in response to cell cycle activation by overexpression of cdk-4 and cyclin D1 (Fig. 8) (17). Similarly, Bernal-Mizrachi and colleagues (46) recently have demonstrated that constitutively active PKB/Akt-1 markedly increases p21 in murine islets. In functional terms, p21 appears to restrain ß-cell replication, because p21–/– islets are significantly more responsive in terms of proliferation to growth factors than normal, p21+/+ islets (17). p21 expression appears to decline in menin-deficient islets as they progressively lose cell cycle restraint (see Section II.J) (61). Interestingly, and in contrast, whereas p21 is increased in response to growth factors and cdk-4/cyclin overexpression, it does not appear to increase in response to metabolic signals such as diabetes induced by a high-fat diet or by leptin receptor deficiency (62). Thus, it is possible that p21 may be a key molecular brake, responding to and counterregulating mitogenic, but not metabolic, signals for ß-cell proliferation.
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). Moreover, they demonstrated that transgenic p27 overexpression under the control of the insulin promoter led to reduced ß-cell proliferation rates, islet hypoplasia, and diabetic ketoacidosis (62). Conversely, Jetton (63) demonstrated that p27 is present in the nucleus of ß-cells in Zucker lean control rats but shifts to the cytoplasm (where it is inactive as a cell cycle inhibitor) in the islets of Zucker fatty rats. These observations make the point that physiological or forced nuclear p27 overexpression reduces ß-cell proliferation and ultimately ß-cell mass and function, whereas translocation to the cytosol is associated with an increase in ß-cell mass. Interestingly, and in contrast to p21, p27 does not appear to be up-regulated in response to mitogenic stimuli such as PL or HGF (17) or constitutively active PKB/Akt-1 (46), suggesting again that p21 may be important in counterregulating mitogenic stimuli, whereas p27 may serve to regulate metabolic stimuli.
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Regarding the third CIP/KIP/WAF member, p57, Kassem et al. (64) have demonstrated that selective or tissue-specific loss of p57 occurs in the proliferating ß-cells of children with the focal variant of hyperinsulinism of infancy. Bushan (65) has suggested that p57 is particularly important embryologically in restraining proliferation of the cells in the primordial pancreatic bud in the mouse but disappears coincident with ß-cell differentiation, as p21 and p27 appear. In contrast, p57 has been deleted (or more accurately, because it is maternally imprinted, deletion of a single allele results in maternally derived, heterozygously null pups that contain a functional biallelic loss), but no islet or metabolic abnormalities have been sought or described (66).
Collectively, these observations suggest that the CIP/KIP/WAF family is centrally important in regulating ß-cell replication, with p57 perhaps being most important embryologically, being replaced by p21 and p27 as the mature ß-cell phenotype is achieved. These findings also suggest that loss of one KIP or CIP or pRb or p53 does not result in loss of cell cycle arrest, but loss of two of these key proteins can.
J. Menin
There is an endocrine tumor syndrome called "multiple endocrine neoplasia type 1" (MEN1), in which affected subjects develop pituitary, parathyroid, and pancreatic islet tumors in an autosomal dominant manner (67). The responsible gene, menin, was recently positionally cloned, but its function has remained poorly defined (67). The menin gene has been knocked out in mice both globally (68) and in a ß-cell-specific manner (69) (Fig. 10, A and B), and these knockouts lead to the development of dramatic ß-cell hyperplasia, islet tumors, hyperinsulinemia, and hypoglycemia. As noted above in the Section II.H, Franklin et al. (56) remarked on the similarity between mice doubly knocked out for p18 and p27 and the MEN syndrome. This suggested to Kim and collaborators (61) that p18 and p27 may be downstream targets of the menin protein, whose loss results in ß-cell hyperplasia and tumors in mice and humans with the MEN type 1 syndrome. Karnick et al. (61) reported recently that mice lacking the MEN-1 syndrome protein, menin, are indeed phenocopies of Franklin’s combined p18- and p27-null mice. They further reported that menin transcriptionally up-regulates the p18 and p27 genes and that its loss results in combined p18 and p27 underexpression (Fig. 10), thus permitting the ß-cell hyperplasia and tumorigenesis that occurs in the MEN1 syndrome (61). Interestingly, as islet hyperplasia progressed, additional INK and KIP loss occurred (Fig. 10), suggesting that additional KIP and INK loss may facilitate further ß-cell replication and that menin may regulate additional KIP and INK members. Finally, the menin story represents a second example in which loss of one inhibitor (e.g., p18 or p27 alone) has little effect on ß-cell mass, whereas combined loss of two cell cycle inhibitors (p18 and p27 or pRb and p53) leads to islet hyperplasia and or insulinoma.
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III. Developing Models of Cell Cycle Control in the ß-Cell |
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can serve as a working model to explore the details of the molecular control of ß-cell replication. However, it is critical to underscore that this model represents only a working model and that such models are frequently oversimplified and/or incorrect. This applies to the ß-cell in particular, as well as to the study of cell cycle control in general. Recently, this has been demonstrated particularly pointedly, with the demonstration that combined global deletion of all three D-cyclins, or of both cdk-4 and cdk-6 (all long believed to be essential for cell cycle progression and control), permit almost complete embryogenesis and organogenesis, including the robust cell division that allows an embryo to go from the one-cell stage to the multimillion-cell stage (70, 71). These reports clearly indicate that we have much to learn regarding normal cell cycle control in every cell type. Furthermore, as emphasized in Section I, as is obvious from the paucity of data in Table 1, and as exemplified by the observation that a protein as important as pRb was not even sought in the islet until 2004 (29), this is particularly true for the study of cell cycle control in the ß-cell, for it remains in its infancy, lagging decades behind that in many other cell types; ß-cell biologists now clearly must catch up with developmental biologists, molecular oncologists, and experts in other types of cells, such as hematopoetic cells, gastrointestinal epithelial cells, fibroblasts, keratinocytes, and yeast. In addition, it is important to point out that although disruption of the G1/S pathway frequently leads to defects in ß-cell replication, this in no way excludes regulatory roles for other cell cycle checkpoints or cell cycle regulatory molecules in ß-cell proliferation. Of course, other cyclins and cyclin-dependent kinases (for example, cyclin B, cdk-1 acting in G2 and mitosis) and their upstream regulatory molecules are essential to the completion of the cell cycle in ß-cells. However, the array of data presented above make the point that the pRb pathway is unquestionably a particularly important control center in the rodent and human ß-cell and strongly suggests that it is an important avenue to explore in any study of the normal or therapeutic regulation of the cell cycle in the ß-cell.
Thus, whereas the studies and models described above represent the current state of the art, they have additional important limitations. One limitation is that studies from our group and others defining the presence and levels of the cell cycle regulatory molecules present in the murine islet, using Western blotting and RT-PCR, have been performed on whole islets, and it is therefore difficult to infer whether changes that are observed—or not observed—apply to the small subpopulations of proliferating ß-cells or are more reflective of the much larger population of arrested cells. More specifically, if a cell cycle event is occurring in 1–3% of cells, this means that it may not be occurring in approximately 97–99% of cells and will likely not be reflected on immunoblots or RT-PCR experiments performed using whole islet extracts. For example, the fact that phospho-pRb is not observed in RIP-HGF, RIP-murine PL-1, or double transgene islets (17) could reflect the fact that pRb is not phosphorylated in response to HGF or murine PL-1, or, in our view more likely, reflects an artifactual "dilution" on Western blots of phospho-pRb in the few (
1–3%) replicating ß-cells by the unphosphorylated pRb in the remaining (97–99%) ß-cells.
Secondly, it is often difficult to know when using immunoblotting or RT-PCR of whole islets how much applies to ß-cells per se, particularly when working with isolated human islets where ß-cells represent a minority (29, 72, 73, 74). This is complicated by the fact that immunohistochemistry and immunoblotting studies are entirely dependent on antibody specificity. Unfortunately, the current antiserum repertoire is inadequate. For example, antibodies for performing studies as fundamental as pRb immunohistochemistry in murine samples are not currently available, nor are antisera that unequivocally differentiate among the murine D-cyclins.
Another limitation is that much cell cycle protein physiology relates not to absolute abundance in the cell, but to subcellular (e.g., nuclear vs. cytoplasmic) localization, as exemplified by the p21 nuclear translocation in Fig. 8 (17), and by the p27 nuclear-cytoplasmic shuttling described by Jetton (63). Moreover, absolute abundance within a particular cellular compartment is not the only important consideration, because even within a given subcellular compartment, other issues are just as critical, such as phosphorylation status, ubiquitination status, and kinase activity, among others. These issues are difficult to study in specific compartments of specific cell types such as ß-cells, and this area therefore remains in its infancy.
It is also critical to define the molecular partners of each of these molecules in a given cell at a given moment in cell cycle progression. For example, cdk-2 and cyclin E may both be present in a cell, but if they are not physically interacting in the nucleus, they will not cause pRb phosphorylation. Or pRb may be present in the nucleus of a ß-cell, but if it does not interact with E2Fs, for example by being competed away by TAg, it will not perform its cell cycle repressive role. Thus, it is essential not only to understand which proteins are present in a given cell, and in which compartments they are present, but also which partners they are currently interacting with and at which phase of the cell cycle such interactions are occurring. This requires coimmunoprecipitation of synchronized cells, but performing these kinds of studies using sufficient quantities of synchronized, purified, and healthy ß-cells is difficult at present.
Another weakness of the current attempts to catalog cell cycle proteins in the ß-cell is that although they may be comprehensive, there are likely not complete. For example, there are many other G1/S regulatory proteins, such as the Id family of basic helix-loop-helix proteins, the DPs, p63, p73, c-Myc, as well as others that are currently unknown; for all we know, these may have centrally important but as yet undefined roles in ß-cell replication (75). Furthermore, cell cycle control proteins such as telomerase may also play critical roles in normal and engineered cell cycle progression (42, 55).
One approach to these hurdles is to try to circumvent them by using mouse genetic studies. These studies often provide powerful information, exemplified by the cdk-4 knockout causing neonatal ß-cell failure and diabetes (Fig. 5) (3, 43), or the cyclin D2 knockout leading to later ß-cell failure and diabetes (Fig. 7) (51, 52), or the menin knockout resulting in combined p18 and p27 loss and cell cycle acceleration in vivo (Fig. 10) (61), or the p27 knockout releasing ß-cell inhibition in response to a high-fat diet (Fig. 9) (62). On the other hand, these models fail in some senses, for they may fail to reveal mechanisms and interacting proteins, or as exemplified by the pRb knockout in embryonic fibroblasts (25), may be completely obscured by compensation by other proteins, in that case, p107. Also, embryonic and neonatal abnormalities, exemplified by the cdk-4 knockout, may obscure later normal physiology in adult ß-cells. Finally, and perhaps most importantly, mouse genetic studies do not always shed faithful light on events in human ß-cells. For example, whereas human children defective or deficient for both alleles of the pRb gene always develop retinoblastomas, knockout of the pRb gene in mice does not cause retinoblastoma (27, 28, 76). And whereas TAg essentially always activates cell cycle progression in rodent cell types, it is not clear that this is true in human tissues.
These limitations can be viewed as obstacles, but they can also be viewed as opportunities for further study in this world of rapidly evolving and expanding commercial antisera inventories, conditional and temporal knockout technologies, and transient and stable small interfering RNA knockdown techniques. Clearly, progress is being made.
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IV. Are We Looking for "Brakes" or "Accelerators"? |
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) are imposed, and cell cycling slows dramatically. Thus, we have come to feel that searching for the molecular accelerator may be missing the point; perhaps we ought to be asking, "what are the molecular brakes, or combinations of brakes, that are so specific for the ß-cell?" "Which of these brakes, alone or in combination, must be removed to allow spontaneous ß-cell replication?" And, "Why are the brakes applied so unusually firmly in the ß-cell, and how do they interfere with therapeutic or compensatory attempts to enhance ß-cell replication?"
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V. Will Cell Cycle Strategies Prove to Be Oncogenic? |
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VI. The Signaling-Cell Cycle Interface |
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. This can only be meaningfully and systematically accomplished when the full spectrum of cell cycle regulatory molecules and their regulatory mechanisms are defined. For example, we know that JAK2-STAT5 pathways are downstream of PRL and PL in the ß-cell and that they up-regulate cyclin D2 (47, 79, 80), but is this the only, or the most important, mechanism for cell cycle activation? We know that HGF activates PI3 kinase (81) and insulin activates insulin receptor substrate-2 signaling (82), but what precisely are the downstream cell cycle molecules that are activated by these signaling molecules, and which of these are most important? One lovely recent example of the complexity of this interface is the demonstration by Bernal-Mizrachi and colleagues (46) that PKB/Akt-1 activation increases in cyclins D1 and D2, cdk-4 kinase activity, and p21, while decreasing p57, the balance of which would be expected to lead to cell cycle progression. Answering these types of questions can be expected to be a growth area in the coming few years. |
VII. Identifying Therapeutic Targets for Driving Cell Cycle Progression |
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Along these same lines, one can expect that intracellular cell cycle target molecules in the G1/S pathway might be natural targets. The challenge here, because the G1/S pathway is so widely represented, will be to develop agents that are sufficiently specific such that they activate the ß-cell cycle without affecting other cell types. Clear precedent for achieving such cell-type specificity exists.
Finally, one must bear in mind that some events or interventions that activate the cell cycle can also activate cell death pathways. One excellent example is that transgenic overexpression of c-Myc in murine islets does indeed drive ß-cell replication, but this is accompanied by marked increases in ß-cell death, the result of which is reduced islet function (75). On the other hand, this is not always the case; for example, adenoviral overexpression of cyclin D1 and cdk-4 results in increases in ß-cell replication unassociated with increases in ß-cell death (29).
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VIII. Summary |
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Footnotes |
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First Published Online April 25, 2006
Abbreviations: cdk, Cyclin-dependent kinase; CIP, cyclin inhibitory protein; HDAC, histone deacetylase; HGF, hepatocyte growth factor; INK, inhibitory kinase; KIP, kinase inhibitory protein; MEF, murine embryonic fibroblast; MEN, multiple endocrine neoplasia; NLS, nuclear localization signal; PI3, phosphatidylinositol 3; PKB, protein kinase B; PL, placental lactogen; PRL, prolactin; PTHrP, PTH-related protein; Rb, retinoblastoma protein; RIP, rat insulin II promoter; TAg, T-antigen.
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