In this study, we sought to test the hypothesis that expression of Id3 in adult human β-cells would repress p57Kip2
expression and induce proliferation. The ability of bHLH transcription factors and Id proteins to regulate cell cycle machinery is well documented,53
and is thought to underlie oncogenic transformation of a variety of cell types.54,55
One mechanism by which Ids promote cell cycle entry is through inhibition of bHLH-mediated expression of the cyclin dependent kinase inhibitors (CDKI).56-58
Conversely, Id knockdown is associated in many cell types with increased expression of CDKIs.53,56,59-63
Recently we reported that E47 directly activates p57Kip2
in a human fetal islet cell line.40
In this study Id3 efficiently suppressed p57Kip2
. Together the data establish that the E47/Id axis controls p57Kip2
levels in human β-cells, similar to observations in human neuroblastoma cells.41
Our results do not support the hypothesis, however, that loss of p57Kip2
is sufficient to induce proliferation in primary adult human β-cells. In the present study, samples were from donors 19 y and older. Because the association between β-cell hyperplasia (PHHI) and p57Kip2
silencing was reported in patients younger than 12 y of age27
it is possible that β-cell replication in response to p57Kip2
downregulation is age dependent. Age related changes in cell cycle genes have been described in murine β-cells.64
Thus, it is possible that loss of p57Kip2
in neonatal human β-cells would trigger cell cycle entry.
In our hands, the β-cell replication stimuli prolactin, exendin-4, did not induce proliferation of control (LacZ-expressing) β-cells. Although there is abundant evidence that these agents increase in vivo islet growth and β-cell mass, as well as in vitro replication of rodent β-cells and insulinoma cell lines,26,43-45
we are unaware of similar data that unequivocally demonstrates a stimulatory effect on human β-cells in vitro. In support of our finding, the in vitro proliferative responses of highly purified rat and human β-cells have been compared.25
The GLP-1 analog liraglutide induced BrdU incorporation in insulin-positive rat but not human β-cells. Furthermore, the result was confirmed when β-cells were identified by PDX-1 staining: no PDX-1+
cells were detected.25
This study is consistent with our finding here that prolactin and exendin-4 are themselves insufficient to induce cell cycle entry in quiescent human β-cells in vitro. Interestingly, we saw a different response under the same conditions with Id3-transduced β-cells. Here, prolactin and exendin-4 synergized with Id3 to increase the percentage of β-cells expressing BrdU. Although the exact explanation remains to be determined, we speculate that prolactin and exendin-4 provide a replication stimulus, but alone it is insufficient to induce cell cycle entry. However, in combination with Id3, these agents are able to increase the proportion of cells attempting DNA synthesis, and subsequently undergoing DNA repair. A similar result was observed following caffeine administration. In caffeine- containing cultures, nearly 100% of insulin + cells were also BrdU-positive, which is consistent with all cells having circumvented the G1
/S checkpoint, only to encounter a downstream block that prevents progression through S phase.
Although we observed BrdU incorporation in β-cells in response to Id3, it did not reflect complete cell cycle progression, as the cells lacked expression of the proliferation markers Ki67, pCyclinE or pHH3. Moreover, the BrdU positive cells exhibited evidence of a DNA damage response. Therefore, the data raise a serious concern about relying solely on BrdU uptake as evidence of β-cell replication because BrdU incorporation can signify either DNA repair or replication. Importantly, the same limitation in interpretation applies to other methods which rely on incorporation of an analog into DNA (e.g., tritiated thymidine or fluorogenic deoxyuridine).65
A recent study of replication marker expression in human β-cells demonstrated that co-expression of two or more markers more accurately discriminates between quiescent and replicating β-cells.66
Expression of Ki67 is used routinely as a proliferation marker in immunohistochemical studies. However, in β-cells, Ki67 is expressed at very low levels through G1
and early in S phase and its expression peaks during late S/G2
Therefore, β-cells which have begun replication but stalled in early S might not be expected to express Ki67. Interestingly, PCNA is also a commonly used proliferation marker,66-70
but its use as a β-cell replication marker has been suggested to overestimate the true replication frequency.70
PCNA is involved in excision-repair,71
and it is tempting to speculate that at least some PCNA staining may also be due to a stimulus-induced DNA repair response, and not replication.
The punctate BrdU staining pattern was an early indicator that replication was not completed in Id3-expressing β-cells because this pattern was distinct from the more homogenous nuclear staining seen in replicating ductal cells (, ) and reference 39
. Moreover, upon close inspection BrdU staining colocalized with DNA repair enzymes. BrdU incorporation that occurs during DNA repair is indicative of double strand breaks and collapsed replication forks.47
The DNA damage response is initiated by recruiting repair enzymes, and shunting of damaged DNA to nuclear pores.48
In undamaged nuclei 53BP1 is diffusely distributed, or is restricted to one or two foci, but relocalizes once phosphorylated. Similarly, histone H2AX is rapidly phosphorylated52
in response to S phase DNA double-strand breaks.51,72
Thus, our observation that γH2AX colocalized with foci containing BrdU and that 53BP1 relocalized in Id3-expressing β-cells is entirely consistent with a repair response. Further evidence for a repair response in Id3 expressing β-cells came from hydroxyurea treatment (HU) which preferentially inhibits BrdU incorporation in replicating cells. Mesenchymal and ductal cells exhibited extreme sensitivity to HU, consistent with DNA replication. In contrast, HU treated β-cells retained BrdU uptake in a large proportion of cells.49
Our demonstration that Id3 expressing β-cells failed to enter the cell cycle was unexpected in the light of our finding that Id3 induces robust cell cycle entry of pancreatic ductal cells.39
Ductal and β-cells are closely related developmentally; ducts give rise to neogenic β-cells during pancreas embryogenesis,73
and we have shown that the process can be recapitulated in the adult human pancreas.5
Therefore, the data appear to reinforce the hypothesis that as β-cells age they lose cell cycle machinery or substrates essential for replication. Consistent with this theory, we recently found that the mitosis protein CENP-A declined with age in humans β-cells, while CENP-A levels remained constant in exocrine cells.74
Further, rodent β-cells did not lose CENP-A to the extent seen in human cells. Such findings may shed light on the context dependent effects of Id3 on β-cells compared with other pancreatic epithelial cell types and between species. The ability of Id3 to induce cell cycle entry and progression in duct cells also served as an important control by ensuring that the Id3 expression levels and conditions employed in these studies were not generally toxic to primary human pancreatic epithelial cells.
A recent study has shown that human islets contain a number of key regulatory molecules necessary for G1
/S transition, including cyclins and cyclin-dependent kinases (cdks).35
Several of these proteins are also differentially expressed between human and rodent β-cells.35,75
Murine β-cells express abundant cdk-4 and cyclin D2, and genetic mouse models have shown them to be critical for β-cell proliferation and diabetes development.76-79
In contrast, human β-cells contain high levels of the analogous enzyme cdk-6, and cyclins D1 and D3 are thought to play a role. Recently, it was reported that overexpression of cdk-6 and cyclin D1 in isolated human β-cells in vitro induced BrdU incorporation and Ki67 expression.35,80
It will be important to determine whether there is concomitant cell cycle completion and an increase in β-cell number as suggested by transplantation studies.
In this study, we found that expression of Id3 in adult human β-cells repressed p57Kip2 expression but did not induce proliferation. Importantly, Id3 mediated efficient cell cycle progression in closely related duct cells thus ruling out adenoviral or Id3 induced general cellular toxicity. The BrdU incorporation and DNA damage markers in β-cells are consistent with a model in which the cells enter S phase in response to Id3, but undergo replication fork stalling due to either an intrinsic limitation in cell cycle machinery or to an abundance of inhibitory factors. Importantly, such questions can be addressed by comparing expression profiles in β-cells vs. ductal epithelial cells. Such studies should lead to a rational strategy for increasing replication competence in adult human β-cells.