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Endocrinology Vol. 138, No. 9 3620-3623
Copyright © 1997 by The Endocrine Society

ARTICLES

Prohormone Convertase 2 Is Necessary for the Formation of Cholecystokinin-22, But Not Cholecystokinin-8, in RIN5F and STC-1 Cells1

Jaeyoung Yoon and Margery C. Beinfeld

Department of Pharmacological and Physiological Science, St. Louis University School of Medicine (J.Y.), St. Louis, Missouri 63104; and the Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine (M.C.B.), Boston, Massachusetts 02111

Address all correspondence and requests for reprints to: Dr. Margery C. Beinfeld, Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111. E-mail: mbeinfel{at}opal.tufts.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two endocrine tumor cell lines from pancreas (RIN5F) and intestine (STC-1) express cholecystokinin (CCK) messenger RNA and are able to posttranslationally process pro-CCK to CCK-22 and CCK-8 amide. Both of these forms are also secreted by these cells. Because they make and secrete forms of amidated CCK larger than CCK-8, they represent a model of pro-CCK processing in the gut and allow investigation of possible mechanisms for tissue differences in prohormone processing.

Both of these cells express two endoproteases convertase-1 (PC1) also known as PC3 and prohormone convertase-2 (PC2), which may be involved in pro-CCK processing. We have previously shown than inhibition of PC1 expression in these cells using stable expression of antisense messenger RNA caused a significant reduction in cellular content of amidated CCK and caused a selective depletion of CCK-8 with a comparative sparing of CCK-22. We demonstrate here that inhibition of PC2 expression in these cells also caused a large initial decrease in CCK content and produced a selective depletion of CCK-22 and a comparative sparing of CCK-8. These results support both a role for both PC1 and PC2 in pro-CCK processing in these cells and the hypothesis that tissue-specific processing of pro-CCK may be explained by differences in expression or activity of PC1 and PC2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHOLECYSTOKININ (CCK) is produced by both endocrine cells and neurons in the gut and by neurons in the brain. After feeding, CCK is released from the duodenum and travels in the circulation to the gall bladder, causing it to contract, and to the pancreas, where it causes the release of digestive enzymes (1, 2). CCK is abundant in the brain, where it serves as a neurotransmitter in a number of important neuronal projections (3).

The brain produces mainly CCK-8 (4), whereas the gut produces larger forms, such as CCK-58, -33, and -22 (5). The tissue differences in forms of CCK are thought to be the result of differential processing of the same prohormone precursor. Thus, the mechanism for tissue differences in processing may lie in the endoproteases.

The discovery and cloning of the Ca2+-dependent subtilisin family of endoproteases have provided a number of good candidates, notably prohormone convertase-1 (PC1) (6), also known as PC3, and PC2 (7). They are widely distributed in neural and endocrine tissue and have been shown to cleave a number of propeptides, including POMC (8, 9), proinsulin (10), proenkephalin (11), proglucagon (12), and prosomatostatin (13). Also, both PC1 and PC2 are expressed in several endocrine cell lines that express CCK messenger RNA (mRNA) and correctly process pro-CCK to amidated products (14).

Other enzymes that may be involved in CCK processing include CCK-8-generating enzyme purified from rat brain synaptosomes (15), which, along with recombinant yeast aspartyl peptidase-3 (16) and PC2 (17), is able to cleave CCK-33 to generate CCK-8.

To examine the possible role of these enzymes in pro-CCK processing, an antisense strategy was employed. This was essential because specific, nontoxic, cell-permeant inhibitors of these enzymes are not available.

The use of antisense message expression in stable cell lines has previously been used to inhibit prohormone convertase expression by Bloomquist et al. to support a role for PC1 in POMC processing (18) in AtT20 cells. Subsequently, it has been used to demonstrate the role of PC2 in proenkephalin (19) and proglucagon processing (12).

In a previous study (20), we inhibited endogenous PC1 expression by stable expression of PC1 antisense mRNA in RIN5F and STC-1 cells, which express CCK mRNA and process pro-CCK to CCK-8 and CCK-22. We demonstrated that expression of PC1 antisense mRNA specifically inhibits CCK-8 formation within these cells. The current study extends our previous work and examines the effect of antisense inhibition of PC2 expression on pro-CCK processing in these same cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of antisense expression plasmids
The antisense PC2 plasmid pCMV5/antiPC2 was constructed using the first 480 bases of the PC2 complementary DNA (cDNA) contained within the prPC2.480EK plasmid provided by Dr. Richard Mains. This PC2 fragment was inserted into pCMV5 (21) at the KpnI and XbaI sites in the antisense orientation. The orientation of the insert was confirmed by restriction mapping.

Maintenance and transfection of tissue culture cells
RIN5F cells and STC-1 cells were maintained as previously described (20) Cells were transfected with expression plasmids by electroporation at 200 V/500 µF using the Bio-Rad Gene Pulser exponential decay-type electroporator (Bio-Rad, Richmond, CA) and subcloned as previously described (20). The pCMV5/antiPC2 was cotransfected with pMtNeo, which confers resistance to the antibiotic G418, in a molar ratio of 5:1.

Western blot analysis
A polyclonal antibody recognizing PC2 proteins was generously provided by Dr. Iris Lindberg, Louisiana State Medical Center (New Orleans, LA) (22, 23). Western analysis was performed as previously described (20). The intensities of proteins bands from the autoradiographs were analyzed by densitometry, using a set area for each lane, with ImageQuant software.

RIA
The CCK RIA was performed as previously described (24), using the rabbit polyclonal CCK antibody (R5) that is specific for amidated forms of CCK. The RIA used [125I]gastrin-17 as tracer, produced by iodination with chloramine-T (25).

Chromatography
Cells from four to eight 10-cm plates were extracted with 0.1 N HCl, pooled, and concentrated by vacuum centrifugation. Cell extracts were neutralized and separated by Sephadex G-50 chromatography in a 35 x 1-cm column run at 4 C in 50 mM Tris and 100 mM NaCl, pH 7.8, containing 0.1% BSA and 0.05% sodium azide. Fractions of 1.0 ml were collected, and aliquots were removed for the CCK RIA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RIN5F and STC-1 cell lines were stably transfected with pCMV5/antiPC2 and selected with G418. CCK levels were determined by RIA and displayed a wide range of values, including some that initially had undetectable CCK levels (Fig. 1Go). After being passaged for several months, the CCK levels of these RIN5F anti-PC2 lines increased enough to allow analysis of their products by gel filtration chromatography.



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  		Figure 1. CCK levels in RIN5F control cells and anti-PC2 cell lines A2C1, A2B6, and A2D7 as a function of time after being established as stable cell lines.

 
A number of STC-1 anti-PC2 cell lines were also obtained. The most severely affected STC-1 cell line, H4, had about 25% of the wild-type CCK levels (Fig. 2Go). This line was selected for further analysis.



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  		Figure 2. CCK levels in control STC-1 cells and anti-PC2 H4 cells.

 
Selected RIN5F and STC-1 clones were analyzed for PC2 expression by Western blotting (Fig. 3Go, A and B) soon after they were established as stable cell lines. PC2 protein was present in 75-, 71-, and 66-kDa forms; of these, the 66-kDa form was the most active (26). The H4 anti-PC2 cell expressed about 50% of the PC2 protein of parent STC-1 cells, D9 control cells (stably transfected with the pCMV5 vector without insert), or the F2 anti-PC1 cell line previous described (20). Among the RIN5F anti-PC2 cell lines, there was a rough correlation between CCK levels and PC2 expression, although some PC2 was detectable even in cells in which amidated CCK was initially undetectable (C1 and D7). Clone D7 had similar PC2 expression as clone C1 (data not shown). In all of these severely affected cell lines, the level of PC2 expression was between 25–50% of that in wild-type RIN5F cells.



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  		Figure 3. PC2 levels by Western analysis. A, Western blot of protein extracts from H4 anti-PC2 STC-1 cells compared with S1F2 (an anti-PC1 cell line), SCC9 (a control cell line transfected with the pCMV5 vector with no insert), and wild-type STC-1 cells. B, Western blot of protein extracts from wild-type RIN5F cells and RIN5F anti-PC2 cells.

 
The effect of expression of PC2 antisense mRNA produced a specific decrease in PC2 protein expression, but it did not alter PC1 or CCK mRNA expression (20).

Sephadex G-50 chromatography and CCK RIA were used to analyze the products of pro-CCK processing of selected clones (Figs. 4Go and 5Go). Chromatography and RIA of cellular extracts of anti-PC2 clones from both RIN5F and STC-1 antisense cell lines compared with those of parental cells indicate that there was a selective depletion of CCK-22 with a comparative sparing of CCK-8.



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  		Figure 4. Sephadex G-50 chromatography of 0.1 N HCl extracts of wild-type STC-1 cells (A) and of STC-1 anti-PC2 H4 cells (B).

 


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  		Figure 5. Sephadex G-50 chromatography of 0.1 N HCl extracts of RIN5F anti-PC2 cell lines B6, C1, and D7 and wild-type RIN5F cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The discovery of the Kex-2 subtilisin family of enzymes has revolutionized the field of prohormone processing. PC1 and PC2 are proving to be very important enzymes in the processing of a wide variety of prohormones. Because specific nontoxic inhibitors have not been developed for these enzymes, the ability to examine their role in prohormone processing in specific cell lines and tissues has been limited. Previous studies (12, 18, 19, 20) have used an antisense strategy, which was also employed in this work.

We show here that stable expression of antisense PC2 mRNA decreases, but does not eliminate, PC2 expression and results in a selective depletion of CCK-22 with a comparative sparing of CCK-8. This is consistent with (but does not prove that) PC2 is essential for the production of amidated CCK-22, but not CCK-8, in STC-1 and RIN5F cells. The effect of the antisense was specific and long lasting. As previously described for RIN5F anti-PC1 cells (20), the anti-PC2 cell lines have been growing continuously for about 1 yr and have not reverted to the wild type in terms of their altered pattern of CCK processing. The reason for the initially low level of CCK content in the anti-PC2 cells and its subsequent rise after being passaged for several months is unknown, but may be due to the initial toxic effect of antibiotic selection or antisense expression, which decreased with time, possibly followed by up-regulation of CCK expression.

The precise sites where PC1 and PC2 cleave are still under investigation. In Fig. 6Go, a model is presented showing where PC1 and PC2 may be acting. It is likely that they are acting directly on pro-CCK itself, but the possibility that either PC1 or PC2 is essential for the activity of another protease that actually cleaves pro-CCK cannot be excluded.



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  		Figure 6. Model of pro-CCK processing. Rat prepro-CCK is shown schematically, with the major amidated forms indicated above the prohormone. Major cleavage sites are indicated by the single letter amino acid abbreviation form. The proposed branched pathway that would generate CCK-22 and CCK-8 independently is indicated below.

 
Our previous results suggest that PC1 is essential for CCK-8 formation in these cells. This observation is further supported by more recent work with At-T20 cells expressing the CCK cDNA (which lack detectable PC2 protein by Western analysis) and processing pro-CCK to CCK-8. Inhibition of PC1 expression in these cells decreases the production of CCK-8, but is not accompanied by the production of new amidated forms (Wang, W., and M. C. Beinfeld, unpublished observations).

The present evidence suggests that PC2 is required for the production of larger amidated peptides such as CCK-22, which are more abundant in gut than in brain. The fact that both of these enzymes appear to be able to cleave pro-CCK at either a single lysine or arginine residue is interesting given that these cleavages had previously been assigned to enzymes that did not have activity at paired basic residue sites (15).

These results support the hypothesis that the differential CCK prohormone processing observed in gut vs. brain can be explained by differences in expression or activity of endoproteases such as PC1 and PC2.


   Acknowledgments
 
We greatly appreciate the gift of the PC2 polyclonal antibody from Dr. Iris Lindberg (Louisiana State Medical Center, New Orleans, LA), the PC2 rat cDNA from Dr. Richard Mains (Johns Hopkins University, Baltimore, MD), and the STC-1 cell line from Dr. Douglas Hanahan (University of California, San Francisco, CA).


   Footnotes
 
1 This work was supported in part by NIH Grants NS-18667 and NS-31602. Back

Received April 4, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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