SUMMARY Mucus production by goblet cells of the large intestine serves as a crucial anti microbial protective mechanism at the interface between the eukaryotic and prokaryotic cells of the mammalian intestinal ecosystem. However, the regulatory pathways involved in goblet cell-induced mucus secretion remain largely unknown. Here we demonstrate that the NLRP6 inflammasome, a recently described regulator of colonic microbiota composition and bio-geographical distribution, is a critical orchestrator of goblet cell mucin granule exocytosis. NLRP6 deficiency leads to defective autophagy in goblet cells and abrogated mucin secretion into the large intestinal lumen. Consequently, NLRP6 inflammasome-deficient mice are unable to clear enteric pathogens from the mucosal surface, rendering them highly susceptible to persistent infection. This study identifies the first innate immune regulatory pathway governing goblet cell mucus secretion, linking non-hematopoietic inflammasome signaling to autophagy and highlighting the goblet cell as a critical innate immune player in the control of intestinal host-microbial mutualism.

Parathyroid hormone-related protein (PTHrP) was discovered as a result of a search for the circulating factor secreted by cancers which causes the common paraneoplastic syndrome humoral hypercalcemia of malignancy. Since the identification of the peptide in 1982 and the cloning of the cDNA in 1987, it has become clear that PTHrP is a prohormone that is posttranslationally cleaved by prohormone convertases to yield a complex family of peptides, each of which is believed to have its own receptor. It is also clear that the PTHrP gene is expressed not only in cancers but also in the vast majority of normal tissues during adult and/or fetal life. In contrast to the situation in humoral hypercalcemia of malignancy in which PTHrP plays the role of a classical "endocrine" hormone, under normal circumstances PTHrP plays predominantly paracrine and/or autocrine roles. These apparent physiological functions are also complex and appear to include 1) regulation of smooth muscle (vascular, intestinal, uterine, bladder) tone, 2) regulation of transepithelial (renal, placental, oviduct, mammary gland) calcium transport, and 3) regulation of tissue and organ development, differentiation, and proliferation. In this review, the discovery of PTHrP, the structure of its gene and its cDNAs, and the posttranslational processing of the initial translation products are briefly reviewed. Attention is then focused on a detailed organ system-oriented review of the normal physiological functions of PTHrP.

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent genetargeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.Parathyroid hormone-related peptide (PTHrP) was initially identified as the tumor product that is responsible for most instances of humoral hypercalcemia of malignancy. In this circumstance, tumors elaborate PTHrP into the systemic circulation in sufficient quantity to cross-react with classical parathyroid hormone (PTH) receptors in bone and kidney, an interaction that provided the initial biochemical clues to the existence of PTHrP and also served as the basis for bioassays that were used in its purification (1-3). PTHrP does not normally appear to circulate, however, so that such effects are of more pathophysiological than physiological relevance (3).

OBJECTIVEInsulin resistance is a major characteristic of type 2 diabetes and is causally associated with obesity. Inflammation plays an important role in obesity-associated insulin resistance, but the underlying mechanism remains unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine with lower circulating levels in obese subjects, and acute treatment with IL-10 prevents lipid-induced insulin resistance. We examined the role of IL-10 in glucose homeostasis using transgenic mice with muscle-specific overexpression of IL-10 (MCK-IL10).RESEARCH DESIGN AND METHODSMCK-IL10 and wild-type mice were fed a high-fat diet (HFD) for 3 weeks, and insulin sensitivity was determined using hyperinsulinemic-euglycemic clamps in conscious mice. Biochemical and molecular analyses were performed in muscle to assess glucose metabolism, insulin signaling, and inflammatory responses.RESULTSMCK-IL10 mice developed with no obvious anomaly and showed increased whole-body insulin sensitivity. After 3 weeks of HFD, MCK-IL10 mice developed comparable obesity to wild-type littermates but remained insulin sensitive in skeletal muscle. This was mostly due to significant increases in glucose metabolism, insulin receptor substrate-1, and Akt activity in muscle. HFD increased macrophage-specific CD68 and F4/80 levels in wild-type muscle that was associated with marked increases in tumor necrosis factor-α, IL-6, and C-C motif chemokine receptor-2 levels. In contrast, MCK-IL10 mice were protected from diet-induced inflammatory response in muscle.CONCLUSIONSThese results demonstrate that IL-10 increases insulin sensitivity and protects skeletal muscle from obesity-associated macrophage infiltration, increases in inflammatory cytokines, and their deleterious effects on insulin signaling and glucose metabolism. Our findings provide novel insights into the role of anti-inflammatory cytokine in the treatment of type 2 diabetes.

Parathyroid hormone–related peptide (PTHrP) appears to play a major role in skeletal development. Targeted disruption of the PTHrP gene in mice causes skeletal dysplasia with accelerated chondrocyte maturation (Amizuka, N., H. Warshawsky, J.E. Henderson, D. Goltzman, and A.C. Karaplis. 1994. J. Cell Biol. 126:1611–1623; Karaplis, A.C., A. Luz, J. Glowacki, R.T. Bronson, V.L.J. Tybulewicz, H.M. Kronenberg, and R.C. Mulligan. 1994. Genes Dev. 8: 277–289). A constitutively active mutant PTH/PTHrP receptor has been found in Jansen-type human metaphyseal chondrodysplasia, a disease characterized by delayed skeletal maturation (Schipani, E., K. Kruse, and H. Jüppner. 1995. Science (Wash. DC). 268:98– 100). The molecular mechanisms by which PTHrP affects this developmental program remain, however, poorly understood. We report here that PTHrP increases the expression of Bcl-2, a protein that controls programmed cell death in several cell types, in growth plate chondrocytes both in vitro and in vivo, leading to delays in their maturation towards hypertrophy and apoptotic cell death. Consequently, overexpression of PTHrP under the control of the collagen II promoter in transgenic mice resulted in marked delays in skeletal development. As anticipated from these results, deletion of the gene encoding Bcl-2 leads to accelerated maturation of chondrocytes and shortening of long bones. Thus, Bcl-2 lies downstream of PTHrP in a pathway that controls chondrocyte maturation and skeletal development.

We report the identification of βIV spectrin, a novel spectrin isolated as an interactor of the receptor tyrosine phosphatase-like protein ICA512. The βIV spectrin gene is located on human and mouse chromosomes 19q13.13 and 7b2, respectively. Alternative splicing of βIV spectrin generates at least four distinct isoforms, numbered βIVΣ1–βIVΣ4 spectrin. The longest isoform (βIVΣ1 spectrin) includes an actin-binding domain, followed by 17 spectrin repeats, a specific domain in which the amino acid sequence ERQES is repeated four times, several putative SH3-binding sites and a pleckstrin homology domain. βIVΣ2 and βIVΣ3 spectrin encompass the NH2- and COOH-terminal halves of βIVΣ1 spectrin, respectively, while βIVΣ4 spectrin lacks the ERQES and the pleckstrin homology domain. Northern blots revealed an abundant expression of βIV spectrin transcripts in brain and pancreatic islets. By immunoblotting, βIVΣ1 spectrin is recognized as a protein of 250 kD. Anti–βIV spectrin antibodies also react with two additional isoforms of 160 and 140 kD. These isoforms differ from βIVΣ1 spectrin in terms of their distribution on subcellular fractionation, detergent extractability, and phosphorylation. In islets, the immunoreactivity for βIV spectrin is more prominent in α than in β cells. In brain, βIV spectrin is enriched in myelinated neurons, where it colocalizes with ankyrinG 480/270-kD at axon initial segments and nodes of Ranvier. Likewise, βIV spectrin is concentrated at the nodes of Ranvier in the rat sciatic nerve. In the rat hippocampus, βIVΣ1 spectrin is detectable from embryonic day 19, concomitantly with the appearance of immunoreactivity at the initial segments. Thus, we suggest that βIVΣ1 spectrin interacts with ankyrinG 480/270-kD and participates in the clustering of voltage-gated Na+ channels and cell-adhesion molecules at initial segments and nodes of Ranvier.

Hepatocyte growth factor (HGF) is produced in pancreatic mesenchyme-derived cells and in islet cells. In vitro, HGF increases the insulin content and proliferation of islets. To study the role of HGF in the islet in vivo, we have developed three lines of transgenic mice overexpressing mHGF using the rat insulin II promoter (RIP). Each RIP-HGF transgenic line displays clear expression of HGF mRNA and protein in the islet. RIPmHGF mice are relatively hypoglycemic in post-prandial and fasting states compared with their normal littermates. They display inappropriate insulin production, striking overexpression of insulin mRNA in the islet, and a 2-fold increase in the insulin content in islet extracts. Importantly, beta cell replication rates in vivo are two to three times higher in RIP-HGF mice. This increase in proliferation results in a 2-3-fold increase in islet mass. Moreover, the islet number per pancreatic area was also increased by approximately 50%. Finally, RIP-mHGF mice show a dramatically attenuated response to the diabetogenic effects of streptozotocin. We conclude that the overexpression of HGF in the islet increases beta cell proliferation, islet number, beta cell mass, and total insulin production in vivo. These combined effects result in mild hypoglycemia and resistance to the diabetogenic effects of streptozotocin. Hepatocyte growth factor (HGF)1 is a mesenchyme-derived protein originally identified as a circulating factor implicated in liver regeneration after hepatic injury or hepatectomy (1-3). It is now recognized that HGF also exhibits its mitogenic, motogenic, and morphogenic activities in a wide variety of cells (4, 5). The active form of HGF is a disulfide-linked heterodimeric protein, which is composed of a 69-kDa ␣-chain and a 34-kDa ␤-chain, containing four kringle domains and a serine protease-like domain, respectively. Active HGF derives from an inactive single chain precursor that is processed and activated by proteolysis. Four proteases have been reported to date to activate HGF in vitro, including blood coagulation factor XIIa, urokinase, tissue-type plasminogen activator, and a serumderived serine protease named HGF activator (6 -9). HGF is primarily a paracrine factor produced by mesenchymal cells that acts on epithelial cells through a membrane-spanning tyrosine kinase receptor, the protein product of the proto-oncogene, c-met (5, 10, 11). The receptor, like the ligand, has a widespread distribution.Messenger RNAs encoding HGF and the HGF receptor, cmet, are highly expressed during the early development of the pancreas, and then maintained at a low level during puberty and adult life (12)(13)(14). HGF has been detected immunohistochemically in the exocrine portion of rabbit pancreas, and in rat and human pancreatic islet cells (15-17). Tissue-type plasminogen activator has been detected in the rat endocrine pancreas, preferentially in somatostatin cells (18). In addition, confocal immunofluorescent studies have preferentially colocalized the c-Met receptor protein to insulin-conta...

The MYC oncogene is frequently mutated and overexpressed in human renal cell carcinoma (RCC). However, there have been no studies on the causative role of MYC or any other oncogene in the initiation or maintenance of kidney tumorigenesis. Here, we show through a conditional transgenic mouse model that the MYC oncogene, but not the RAS oncogene, initiates and maintains RCC. Desorption electrospray ionization-mass-spectrometric imaging was used to obtain chemical maps of metabolites and lipids in the mouse RCC samples. Gene expression analysis revealed that the mouse tumors mimicked human RCC. The data suggested that MYC-induced RCC up-regulated the glutaminolytic pathway instead of the glycolytic pathway. The pharmacologic inhibition of glutamine metabolism with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide impeded MYC-mediated RCC tumor progression. Our studies demonstrate that MYC overexpression causes RCC and points to the inhibition of glutamine metabolism as a potential therapeutic approach for the treatment of this disease.MYC oncogene | renal cell carcinoma | desorption electrospray ionization mass spectrometry imaging | glutamine metabolism R enal cell adenocarcinoma (RCC) is a kidney cancer that originates in the lining of the proximal convoluted tubule, a part of the very small tubes in the kidney that transport waste molecules from the blood to the urine. Most patients who present with advanced RCC have a dismal prognosis because RCC easily metastasizes and advances in therapy have been limited (1-3). A lack of transgenic models of RCC has made it difficult to identify and test new therapeutic modalities.The MYC pathway is activated in most cases of human RCC (4), genomically amplified in 5-10% of patients, overexpressed in 20% (5), and associated with a hereditary RCC syndrome (6) suggesting a causal role in the pathogenesis, but this has never been examined. Here, we report the development of a conditional transgenic mouse model for MYC-deregulated human RCC. The MYC oncogene contributes to tumorigenesis of many types of cancer through various mechanisms (7-10), including the regulation of proliferation and growth, protein and ribosomal biogenesis, changes in metabolism, lipid synthesis, and induction of angiogenesis (11)(12)(13)(14). MYC reprogramming can result in tumors that are addicted to glucose and/or glutamine for their energy metabolism (15-19). MYC directly regulates specific genes of the glycolytic and glutaminolytic pathways (15,17,20,21), including lactate dehydrogenase A (LDHA), glucose transporter 1 (Glut1), hexokinase 2 (HK2), phosphofructokinase-M 1 (PFKM1), and enolase 1 (Eno1) (21-23). Also, MYC coordinates genes involved in glutamine catabolism (SI MYC and Glutamine Catabolism). However, there has been no evidence to show that MYC overexpression directly drives and maintains RCC or how this occurs.Through our new transgenic mouse model, we showed that transgenic MYC, but not mutant RAS, overexpression in vivo rapidly initiates a highly aggressive RCC that histologi...