Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects

Abstract

The subtilisin-like proprotein convertases PC1/3 (SPC3) and PC2 (SPC2) are believed to be the major endoproteolytic processing enzymes of the regulated secretory pathway. They are expressed together or separately in neuroendocrine cells throughout the brain and dispersed endocrine system in both vertebrates and invertebrates. Disruption of the gene-encoding mouse PC1/3 has now been accomplished and results in a syndrome of severe postnatal growth impairment and multiple defects in processing many hormone precursors, including hypothalamic growth hormone-releasing hormone (GHRH), pituitary proopiomelanocortin to adrenocorticotropic hormone, islet proinsulin to insulin and intestinal proglucagon to glucagon-like peptide-1 and -2. Mice lacking PC1/3 are normal at birth, but fail to grow normally and are about 60% of normal size at 10 weeks. They lack mature GHRH, have low pituitary growth hormone (GH) and hepatic insulin-like growth factor-1 mRNA levels and resemble phenotypically the "little" mouse (Gaylinn, B. D., Dealmeida, V. I., Lyons, C. E., Jr., Wu, K. C., Mayo, K. E. & Thorner, M. O. (1999) Endocrinology 140, 5066-5074) that has a mutant GHRH receptor. Despite a severe defect in pituitary proopiomelanocortin processing to mature adrenocorticotropic hormone, blood corticosterone levels are essentially normal. There is marked hyperproinsulinemia but without impairment of glucose tolerance. In contrast, PC2-null mice lack mature glucagon and are chronically hypoglycemic (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H. & Steiner, D. (1997) Proc. Natl. Acad. Sci. USA 94, 6646-6651). The PC1/3-null mice differ from a human subject reported with compound heterozygosity for defects in this gene, who was of normal stature but markedly obese from early life. The PC1/3-null mice are not obese. The basis for these phenotypic differences is an interesting topic for further study. These findings prove the importance of PC1/3 as a key neuroendocrine convertase.

Fig 1.

PC1/3-null mice exhibit growth retardation and display abnormal growth-related hormone levels. (A) The growth curve of PC1/3-null mice (18 −/−, 20 +/−, and 17 +/+ males were measured). (Inset) One example of body size difference between wt (Left) and null mice at 6 weeks of age. (B) GH mRNA level is decreased in null-mouse pituitary. Northern blot analysis of pituitary GH mRNA was quantified by PhosphorImager (Cyclone, Packard). The loading was normalized against GAPDH. n = 3. (C) GH staining in anterior pituitary lobe reveals smaller somatotrophs with shrunken nuclei in nulls (magnification: ×100). (D) Liver IGF-1 mRNA is decreased in PC1/3-null mice. A Northern blot was analyzed as described in B. n = 2. (E) Circulating IGF-1 is decreased in null mice. n = 3.

Fig 2.

GH-releasing hormone processing is impaired in PC1/3-null mice. (A) Schematic representation of proGHRH (29). Arrowheads indicate sites of proteolytic processing, which are essential for the conversion of proGHRH to mature GHRH. (B) Gel filtration profiles demonstrate shift of GHRH immunoreactivity to the position of proGHRH in PC1/3-null mice.

Fig 3.

POMC processing is impaired in PC1/3-null mice. (A) Schematic representation of POMC processing by PC1/3 and PC2 (5 and 6). Partially processed sites are indicated by small arrows. JP, joining peptide; LPH, lipotropin; endo, endorphin; MSH, melanocyte-stimulating hormone. (B) Western blot shows elevated whole pituitary POMC level in PC1/3-null mice. Samples are arranged in successive three-fold serial dilution (L→R), starting with 0.4 pituitary equivalents (1×). W, wild type; N, null. (C) POMC mRNA is up-regulated in pituitaries of PC1/3-null mice. Samples were prepared and analyzed as in Fig. 1B, n = 3. (D) ACTH is absent in PC1/3-null mice. Anterior pituitaries were labeled as described in Materials and Methods. Samples were immunoprecipitated with antiserum JH93 and size-separated by using a borate-SDS/PAGE tube-gel system. The gels were sliced, and the radioactivity in each slice was determined by scintillation counting. (E) ACTH RIA of whole pituitary extracts resolved by gel permeation chromatography. Antiserum specific to the C-terminal portion of ACTH was used to estimate levels of ACTH-ir peptides in PC1/3-null and wt pituitaries. No intact ACTH was observed in the PC1/3 null; this product is normally generated by PC1/3 in the anterior lobe. However, PC1/3 nulls contain a smaller ACTH-derived peptide (CLIP), arising mainly from PC2 action in the pituitary intermediate lobe.

Fig 4.

Intraperitoneal glucose tolerance test. The tests were performed on fasted animals 16 weeks of age as described in Materials and Methods.

Fig 5.

GLP-1 and GLP-2 are not detectable in the PC1/3-null mice. (Top) Schematic of proglucagon indicating sites of processing by PC2 and PC1/3 (1, 3, 4). Samples from wt and null mice small intestines were extracted and fractionated. GLP-1 and GLP-2 in each fraction were determined by RIA. The readings from GLP-containing fractions were pooled as depicted in the bar graphs. GLP-1 immunoreactivity was measured by using (A) antiserum 2135, an antiserum that measures all GLP-1-containing forms, or (B) antiserum 93242, which is specific for the NH₂ terminus of GLP_7–37 (tGLP) and reacts minimally with N-terminally extended forms of GLP-1. GLP-2 immunoreactivity was measured by using (C) antiserum 312–01, which measures all GLP-2-containing forms, or (D) antiserum 92160, which is specific for N-terminal GLP-2 and reacts minimally with N-terminally extended or truncated forms of GLP-2.