Research Article: Cdk4 Regulates Recruitment of Quiescent β-Cells and Ductal Epithelial Progenitors to Reconstitute β-Cell Mass

Date Published: January 13, 2010

Publisher: Public Library of Science

Author(s): Ji-Hyeon Lee, Junghyo Jo, Anandwardhan A. Hardikar, Vipul Periwal, Sushil G. Rane, Kathrin Maedler.

Abstract: Insulin-producing pancreatic islet β cells (β-cells) are destroyed, severely depleted or functionally impaired in diabetes. Therefore, replacing functional β-cell mass would advance clinical diabetes management. We have previously demonstrated the importance of Cdk4 in regulating β-cell mass. Cdk4-deficient mice display β-cell hypoplasia and develop diabetes, whereas β-cell hyperplasia is observed in mice expressing an active Cdk4R24C kinase. While β-cell replication appears to be the primary mechanism responsible for β-cell mass increase, considerable evidence also supports a contribution from the pancreatic ductal epithelium in generation of new β-cells. Further, while it is believed that majority of β-cells are in a state of ‘dormancy’, it is unclear if and to what extent the quiescent cells can be coaxed to participate in the β-cell regenerative response. Here, we address these queries using a model of partial pancreatectomy (PX) in Cdk4 mutant mice. To investigate the kinetics of the regeneration process precisely, we performed DNA analog-based lineage-tracing studies followed by mathematical modeling. Within a week after PX, we observed considerable proliferation of islet β-cells and ductal epithelial cells. Interestingly, the mathematical model showed that recruitment of quiescent cells into the active cell cycle promotes β-cell mass reconstitution in the Cdk4R24C pancreas. Moreover, within 24–48 hours post-PX, ductal epithelial cells expressing the transcription factor Pdx-1 dramatically increased. We also detected insulin-positive cells in the ductal epithelium along with a significant increase of islet-like cell clusters in the Cdk4R24C pancreas. We conclude that Cdk4 not only promotes β-cell replication, but also facilitates the activation of β-cell progenitors in the ductal epithelium. In addition, we show that Cdk4 controls β-cell mass by recruiting quiescent cells to enter the cell cycle. Comparing the contribution of cell proliferation and islet-like clusters to the total increase in insulin-positive cells suggests a hitherto uncharacterized large non-proliferative contribution.

Partial Text: Pancreatic β-cells are uniquely endowed with the ability to synthesize and secrete insulin – a hormone essential for glucose control [1]. Autoimmune destruction of β-cells results in Type 1 diabetes. Type 2 diabetes is characterized by significantly reduced β-cell mass that combines with β-cell dysfunction resulting in a deficit in β-cell compensation mechanisms in the face of glucose intolerance and insulin resistance [2], [3], [4]. Therefore, restoration of β-cell mass is of major clinical significance in both forms of diabetes. It is known that adult β-cells exhibit limited proliferation capacity that is dependent on genetic background [5], [6], [7]. Furthermore, β-cells turn over slowly and their proliferation potential decreases with age [8], [9]. Several potential mechanisms for regulating β-cell mass have been supported by ongoing research [10]. Pancreatic stem cells, embryonic or arising from diverse locations such as pancreatic ducts, islets and bone marrow, have been proposed as sources of insulin-producing β-cells [11], [12], [13], [14], [15], [16]. Other reported sources are trans-differentiation of pancreatic acinar cells, liver cells, differentiation of intra-islet precursors or splenocytes, and epithelial-mesenchymal transition [17], [18], [19], [20], [21], [22], [23], although recent studies have challenged some of these findings [24], [25], [26]. Furthermore, induced genetic reprogramming of adult exocrine cells to functional β-cells has been recently reported [27]. Among these possible sources, elegant lineage tracing analyses and other approaches convincingly demonstrate that β-cell self-duplication is a dominant source of adult β-cells [28], [29], [30]. A recent report shows the existence of facultative stem cells in the pancreatic ductal epithelium and their recruitment in response to an acute pancreatic injury [31]. These results suggest that the two major mechanisms that increase β-cell mass are (i) duplication of pre-existing β-cells and (ii) generation of β-cells via recruitment of facultative stem/progenitor cells within the pancreatic ductal epithelium.

β-cells, like all other cells, are under the regulatory checks and balances enforced by changes in cell cycle progression [33]. Proof of this concept was provided using mouse models wherein the locus that codes for Cdk4, one of the important gate-keepers of the mammalian cell cycle machinery, was targeted by genetic recombineering [36], [53]. These studies illustrated that loss of Cdk4 results in β-cell hypoplasia and diabetes. In contrast, inheritance of a mutant Cdk4R24C kinase, that is refractory to cell cycle inhibition by p16Ink4a, resulted in β-cell hyperplasia [35], [36]. Although these studies illustrated the unique role of Cdk4 in β-cell mass regulation, the mechanisms by which Cdk4 controls β-cell mass have remained unclear. Here, using a model of partial pancreatectomy, we showed that Cdk4 regulates post-natal β-cell mass by targeting two different cellular compartments: the pre-existing β-cells within islets and presumptive progenitors localized in the pancreatic ductal epithelium. Furthermore, we show that Cdk4 utilizes two distinct mechanisms to engineer β-cell mass re-constitution in response to pancreatectomy. First, the data is compatible with Cdk4 promotion of islet β-cell replication, a mechanism that is presumed to play a primary role in regulating β-cell mass. Second, Cdk4 catalyzes the recruitment of quiescent cells within the islets and the ductal epithelium to participate in the regenerative process. Interestingly, recruitment of quiescent cells within the ductal epithelium displays hallmarks of early pancreatic development [12]. Therefore, pancreatectomy induces rapid proliferation of ductal epithelial cells and a coincident significant increase in cells that express Pdx-1, a transcription factor that is involved in the earliest stages of embryonic pancreas development. The rapid proliferation kinetics combined with the appearance of Pdx-1+ cells suggests involvement of activated progenitors in response to injury. Moreover, we observe increased insulin-positive cells in the ductal epithelium and a significant enhancement in the number of duct-associated small ICCs. Together, we illustrate that Cdk4 stimulates β-cell regeneration by promoting β-cell replication and inducing progenitor cell activation by recruitment of quiescent cells into the active cell cycle (Fig. 7).



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