In the regulated secretory pathway, secretory granules (SGs) store peptide hormones that are released on demand. SGs are formed at the trans-Golgi network and must undergo a maturation process to become responsive to secretagogues. The production of mature SGs requires concentrating newly synthesized soluble content proteins in granules whose membranes contain the appropriate integral membrane proteins. The mechanisms underlying the sorting of soluble and integral membrane proteins destined for SGs from other proteins are not yet well understood. For soluble proteins, luminal pH and divalent metals can affect aggregation and interaction with surrounding membranes. The trafficking of granule membrane proteins can be controlled by both luminal and cytosolic factors. Cytosolic adaptor proteins (APs), which recognize the cytosolic domains of proteins that span the SG membrane, have been shown to play essential roles in the assembly of functional SGs. Adaptor protein 1A (AP-1A) is known to interact with specific motifs in its cargo proteins and with the clathrin heavy chain, contributing to the formation of a clathrin coat. AP-1A is present in patches on immature SG membranes, where it removes cargo and facilitates SG maturation. AP-1A recruitment to membranes can be modulated by Phosphofurin Acidic Cluster Sorting protein 1 (PACS-1), a cytosolic protein which interacts with both AP-1A and cargo that has been phosphorylated by casein kinase II. A cargo/PACS-1/AP-1A complex is necessary to drive the appropriate transport of several cargo proteins within the regulated secretory pathway. The Golgi-localized, γ-ear containing, ADP-ribosylation factor binding (GGA) family of APs serve a similar role. We review the functions of AP-1A, PACS-1, and GGAs in facilitating the retrieval of proteins from immature SGs and review examples of cargo proteins whose trafficking within the regulated secretory pathway is governed by APs.

The adaptor protein 1A complex (AP-1A) transports cargo between the trans-Golgi network (TGN) and endosomes. In professional secretory cells, AP-1A also retrieves material from immature secretory granules (SGs). The role of AP-1A in SG biogenesis was explored using AtT-20 corticotrope tumor cells expressing reduced levels of the AP-1A μ1A subunit. A two-fold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non-condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue-stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α-amidating monooxygenase-1 (PAM-1), integral membrane enzymes that enter immature SGs. The non-condensing SGs contained POMC products and PAM-1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM-1 into PHM was unaltered, but PHM basal secretion was increased in sh-μ1A PAM-1 cells. Despite lacking a canonical AP-1A binding motif, yeast two-hybrid studies demonstrated an interaction between the PAM-1 cytosolic domain and AP-1A. Co-immunoprecipitation experiments with PAM-1 mutants revealed an influence of the luminal domains of PAM-1 on this interaction. Thus, AP-1A is crucial for normal SG biogenesis, function and composition.

Proteins involved in iron homeostasis have been identified as biomarkers for lupus nephritis, a serious complication of systemic lupus erythematosus (SLE). We tested the hypothesis that renal iron accumulation occurs and contributes to renal injury in SLE. Renal non-heme iron levels were increased in the (New Zealand Black x New Zealand White) F1 (NZB/W) mouse model of lupus nephritis compared with healthy New Zealand White (NZW) mice in an age- and strain-dependent manner. Biodistribution studies revealed increased transferrin-bound iron accumulation in the kidneys of albuminuric NZB/W mice, but no difference in the accumulation of non-transferrin bound iron or ferritin. Transferrin excretion was significantly increased in albuminuric NZB/W mice, indicating enhanced tubular exposure and potential for enhanced tubular uptake following filtration. Expression of transferrin receptor and 24p3R were reduced in tubules from NZB/W compared to NZW mice, while ferroportin expression was unchanged and ferritin expression increased, consistent with increased iron accumulation and compensatory downregulation of uptake pathways. Treatment of NZB/W mice with the iron chelator deferiprone significantly delayed the onset of albuminuria and reduced blood urea nitrogen concentrations. Together, these findings suggest that pathological changes in renal iron homeostasis occurs in lupus nephritis, contributing to the development of kidney injury.

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Prostaglandin E 2 (PGE 2 ) is reported to play an important role in tumor development. We explored the differential expression of genes governing production of, and response to, PGE 2 during development of invasive bladder cancer. N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) or vehicletreated mice (n = 4-5) were euthanized after 4-8 weeks (period 1, P1), 12-16 weeks (P2), and 20-23 weeks (P3). Half of each bladder was analyzed histologically and the other half extracted for mRNA analysis by quantitative real-time PCR. Bladders from BBN-treated mice showed progression from submucosal inflammation (P1) to squamous metaplasia/focal CIS (P2) to poorly differentiated, invasive cancer (P3). mRNA levels for the inducible cyclooxygenase, COX-2, were elevated three to fourfold at all time points in BBN-treated mice compared to controls. In contrast, mRNA levels for constitutive COX-1 and cytosolic phospholipase A 2 (cPLA 2 ), which releases substrate for COX, were either unchanged or decreased in BBN-treated mice relative to controls. Downstream of COX, mRNA levels of membrane-bound PGE 2 synthase (mPGES-1) were increased 1.7-fold at P1 in BBN bladders but returned to control levels at P2 and P3. mRNA levels for 15-prostaglandin dehydrogenase (PGDH), which inactivates PGE 2 , were reduced 50-80% in BBN-treated bladders at all time points. mRNA levels for EP2R and EP4R, receptors for PGE 2 , were two to threefold increased at P1, but returned to control levels or below at P3. Hence, increased COX-2 and decreased PDGH expression occurred throughout tumor development, while mPGES-1, EP2R and EP4R were elevated only before development of invasive cancer. We compared expression of these genes in the malignant human urothelial cell lines, HTB-5 and HT-1376, with expression in a benign urothelial cell line, UROtsa. Neither malignant cell line reproduced the complete in vivo pattern, relative to benign cells, but each showed abnormal basal expression of several of the genes downstream of COX-2, but not COX-2 itself. We conclude that components involved in PGE 2 synthesis and activity are differentially regulated during bladder tumor development and the therapeutic efficacy of targeting the various components may vary with stage of tumor development.

Systemic lupus erythematosus (SLE) is an autoimmune disease predominantly affecting women and often leading to lupus nephritis and kidney damage. Endoplasmic reticulum (ER) stress has been implicated in several forms of kidney disease, but whether ER stress contributes to renal injury in SLE is unknown. To investigate this, a small molecule chaperone, sodium 4-phenylbutyrate (4-PBA), was administered to the New Zealand Black x New Zealand White F1 hybrid (NZBWF1) mouse model of SLE. In a prevention study, treatment with 4-PBA from 20 weeks of age (prior to the development of renal injury) delayed the onset of albuminuria and significantly reduced additional indices of renal injury compared with vehicle-treated NZBWF1 mice at 36 weeks of age, including collagen deposition, tubular casts, renal cell apoptosis, and blood urea nitrogen (BUN) concentration. To test whether ER stress contributes to the progression of renal injury once albuminuria has developed, mice were monitored for the onset of albuminuria (3+ or ≥300 mg/dl by dipstick measurement of 24-h urine sample) and once established, were either killed (onset group), or underwent 4-PBA or vehicle treatment for 4 weeks. Treatment with 4-PBA blocked the worsening of glomerular injury, reduced the number of dilated or cast-filled tubules, and reduced the number of apoptotic cells compared with vehicle-treated mice. BUN and left ventricle to bodyweight ratio (LV:BW) were also reduced by 4-PBA treatment. Renal expression of the endogenous chaperones, protein disulphide isomerase (PDI), and 78 kDa glucose-regulated protein (GRP78, also known as binding Ig protein (BiP)), were increased in 4-PBA-treated mice. Together, these results suggest a therapeutic potential for agents like 4-PBA in combating renal injury in SLE.

The anterior pituitary is specialized for the synthesis, storage and release of peptide hormones. The activation of inactive peptide hormone precursors requires a specific set of proteases and other post-translational processing enzymes. High levels of peptidylglycine α-amidating monooxygenase (PAM), an essential peptide processing enzyme, occur in the anterior pituitary. PAM, which converts glycine-extended peptides into amidated products, requires copper and zinc to support its two catalytic activities and calcium for structure. We used X-ray fluorescence microscopy on rat pituitary sections and inductively coupled plasma mass spectrometry on subcellular fractions prepared from rat anterior pituitary to localize and quantify copper, zinc and calcium. X-ray fluorescence microscopy indicated that the calcium concentration in pituitary tissue was about 2.5 mM, 10-times more than zinc and 50-times more than copper. Although no higher than cytosolic levels, secretory granule levels of copper exceeded PAM levels by a factor of 10. Atp7a, which transports copper into the lumen of the secretory pathway, was enriched in endosomes and Golgi, not in secretory granules. If Atp7a transfers copper directly to PAM, this pH-dependent process is likely to occur in endosomes.