John M. Schmitt

Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation

Hormonal stimulation of cyclic adenosine monophosphate (cAMP) and the cAMP-dependent protein kinase PKA regulates cell growth by multiple mechanisms. A hallmark of cAMP is its ability to stimulate cell growth in many cell types while inhibiting cell growth in others. In this review, the cell type-specific effects of cAMP on the mitogen-activated protein (MAP) kinase (also called extracellular signal-regulated kinase, or ERK) cascade and cell proliferation are examined. Two basic themes are discussed. First, the capacity of cAMP for either positive or negative regulation of the ERK cascade accounts for many of the cell type-specific actions of cAMP on cell proliferation. Second, there are several specific mechanisms involved in the inhibition or activation of ERKs by cAMP. Emerging new data suggest that one of these mechanisms might involve the activation of the GTPase Rap1, which can activate or inhibit ERK signaling in a cell-specific manner.

PKA Phosphorylation of Src Mediates cAMP's Inhibition of Cell Growth via Rap1

In fibroblast cells, cAMP antagonizes growth factor activation of ERKs and cell growth via PKA and the small G protein Rap1. We demonstrate here that PKAs activation of Rap1 was mediated by the Rap1 guanine nucleotide exchange factor C3G, the adaptor Crk-L, the scaffold protein Cbl, and the tyrosine kinase Src. Src was required for cAMP activation of Rap1 and the inhibition of ERKs and cell growth. PKA activated Src both in vitro and in vivo by phosphorylating Src on serine 17 within its amino terminus. This phosphorylation was required for cAMPs activation of Src and Rap1, as well as cAMPs inhibition of ERKs and cell proliferation. This study identifies an antiproliferative role for Src in the physiological regulation of cell growth by cAMP.

Recombinant human bone morphogenetic protein‐2 and collagen for bone regeneration

The study reported describes a combination of recombinant human bone morphogenetic protein-2 (rhBMP-2) and collagen (C) to regenerate bone. Unilateral critical-sized defects (CSDs) were prepared in radii of 32 skeletally mature New Zealand white rabbits. Rabbits were divided evenly among four treatments: autograft, absorbable C (Helistat), 35 microg of rhBMP-2 combined with absorbable C (rhBMP-2/C), and untreated CSDs. The two euthanasia periods were 4 and 8 weeks. Radiographs were taken the day of surgery, every 2 weeks, and at term and the percent of radiopacity was measured. Data analysis revealed a time-dependent increase in the percent radiopacity with rhBMP-2/C. Histological examination revealed the rhBMP-2/C treatment regenerated osseous contour by 8 weeks. According to quantitative histomorphometry, the CSD and C groups had significantly less new bone than either autograft or rhBMP-2/C (p < or = 0.05). The results suggest that rhBMP-2/C could be an effective therapy to restore segmental bone defects.

β2-Adrenergic Receptor Activates Extracellular Signal-regulated Kinases (ERKs) via the Small G Protein Rap1 and the Serine/Threonine Kinase B-Raf

G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade. Heterotrimeric G proteins are composed of both α and βγ subunits that can signal independently to diverse intracellular signaling pathways including those that activate MAP kinases. In this study, we examined the ability of isoproterenol, an agonist of the β2-adrenergic receptor (β2AR), to stimulate extracellular signal-regulated kinases (ERKs). Using HEK293 cells, which express endogenous β2AR, we show that isoproterenol stimulates ERKs via β2AR. This action of isoproterenol requires cAMP-dependent protein kinase and is insensitive to pertussis toxin, suggesting that Gαs activation of cAMP-dependent protein kinase is required. Interestingly, β2AR activates both the small G proteins Rap1 and Ras, but only Rap1 is capable of coupling to Raf isoforms. β2AR inhibits the Ras-dependent activation of both Raf isoforms Raf-1 and B-Raf, whereas Rap1 activation by isoproterenol recruits and activates B-Raf. β2AR activation of ERKs is not blocked by expression of RasN17, an interfering mutant of Ras, but is blocked by expression of either RapN17 or Rap1GAP1, both of which interfere with Rap1 signaling. We propose that isoproterenol can activate ERKs via Rap1 and B-Raf in these cells.

Cyclic AMP-mediated inhibition of cell growth requires the small G protein Rap1

ABSTRACT In many normal and transformed cell types, the intracellular second messenger cyclic AMP (cAMP) blocks the effects of growth factors and serum on mitogenesis, proliferation, and cell cycle progression. cAMP exerts these growth-inhibitory effects via inhibition of the mitogen-activated protein (MAP) kinase cascade. Here, using Hek293 and NIH 3T3 cells, we show that cAMPs inhibition of the MAP kinase cascade is mediated by the small G protein Rap1. Activation of Rap1 by cAMP induces the association of Rap1 with Raf-1 and limits Ras-dependent activation of ERK. In NIH 3T3 cells, Rap1 is required not only for cAMPs inhibition of ERK activation but for inhibition of cell proliferation and mitogenesis as well.

Impact of nicotine on bone healing.

A limited number of experimental animal studies and in vitro data confirm that nicotine impairs bone healing, diminishes osteoblast function, causes autogenous bone graft morbidity, and decreases graft biomechanical properties. Therefore, our long-term goal is to develop an effective therapy to reverse the adverse impact of nicotine from tobacco products. However, before accomplishing this goal, we had to develop an animal model. Our hypotheses were nicotine administration preceding and following autogenous bone grafting adversely affected autograft incorporation and depressed donor site healing in a characterized animal wound model. Hypothesis testing was accomplished in bilateral, 4-mm diameter parietal bone defects prepared in 60 Long-Evans rats (male, 35-day-old). A 4-mm diameter disk of donor bone was removed from the left parietal bone and placed in the contralateral defect. The donor site served as a spontaneously healing bone wound. The rats were partitioned equally among three doses of nicotine administered orally in the drinking water (12.5, 25, and 50 mg/L). For each dose, the duration and sequence of nicotine treatment followed four courses, including no nicotine and designated combinations of nicotine administration and abatement prior to and following osseous surgery. Experimental sites were recovered on 14 and 28 days postsurgery, responses quantitated, and data analyzed by analysis of variance and post hoc statistics (p < or = 0.05). We developed a convenient and effective osseous model, and the results validated our hypothesis that nicotine negatively impacts on bone healing.

CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Activated Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1

ABSTRACT Proliferation of T cells via activation of the T-cell receptor (TCR) requires concurrent engagement of accessory costimulatory molecules to achieve full activation. The best-studied costimulatory molecule, CD28, achieves these effects, in part, by augmenting signals from the TCR to the mitogen-activated protein (MAP) kinase cascade. We show here that TCR-mediated stimulation of MAP kinase extracellular-signal-regulated kinases (ERKs) is limited by activation of the Ras antagonist Rap1. CD28 increases ERK signaling by blocking Rap1 action. CD28 inhibits Rap1 activation because it selectively stimulates an extrinsic Rap1 GTPase activity. The ability of CD28 to stimulate Rap1 GTPase activity was dependent on the tyrosine kinase Lck. Our results suggest that CD28-mediated Rap1 GTPase-activating protein activation can help explain the augmentation of ERKs during CD28 costimulation.

Tissue-engineered bone biomimetic to regenerate calvarial critical- sized defects in athymic rats

A tissue-engineered bone biomimetic device was developed to regenerate calvaria critical-sized defects (CSDs) in athymic rats. Well-documented evidence clearly confirms that left untreated, CSDs will not spontaneously regenerate bone. To accomplish regeneration, four candidate treatments were assessed: porous poly(D,L-lactide) and type I collagen (PLC), PLC and human osteoblast precursor cells (OPCs) at 2 x 10(5) (PLC/OPCs), PLC and 50 microg of recombinant human bone morphogenetic protein-2 (PLC/rhBMP-2), and PLC/OPCs/rhBMP-2 (the bone biomimetic device). The hypotheses for this study were PLC/OPCs/rhBMP-2 would promote more new bone formation in CSDs than the other treatments and the amount of bone formation would be time dependent. To test the hypotheses, outcomes from treatments were measured at 2 and 4 weeks postoperatively by radiomorphometry for percent radiopacity and by histomorphometry for square millimeters of new bone formation. Data were analyzed by analysis of variance and Fishers protected least significant difference for multiple comparisons with p < or = 0.05. At 2 and 4 weeks, radiomorphometric data revealed PLC/rhBMP-2 and PLC/OPCs/rhBMP-2 promoted significantly more radiopacity than either PLC or PLC/OPCs. Histomorphometry data at 2 and 4 weeks indicated significantly more new bone formation for PLC/rhBMP-2, PLC/OPCs/rhBMP-2, and PLC/OPCs compared to PLC. By 4 weeks, PLC/OPCs/rhBMP-2 and PLC/rhBMP-2 had regenerated the CSDs with more new bone than the other treatments; the quantity of bone at 4 weeks for these treatments was greater than at 2 weeks.

Radiomorphometry and Biomechanical Assessment of Recombinant Human Bone Morphogenetic Protein 2 and Polymer in Rabbit Radius Ostectomy Model

The study objective was to determine the mechanical integrity and radiopacity of regenerated bone within critical-sized defects (CSDs) in radii of rabbits using recombinant human bone morphogenetic protein 2 (rhBMP-2) with a porous, biodegradable poly(D,L-lactic acid) (PDLLA) carrier (designated PLA). Twenty millimeter, unilateral radial ostectomies were created in 96 skeletally mature New Zealand white rabbits. The rabbits were randomly assigned to six treatment groups with two euthanasia periods. Treatment groups included unfilled defect (n = 8), segmental autograft (n = 8), PLA + 0 microg rhBMP-2 (n = 8), PLA + 17 microg rhBMP-2 (n = 8), PLA + 35 microg rhBMP-2 (n = 8), and PLA + 70 microg rhBMP-2 (n = 8). The radiopacity was significantly greater for the 35- and 70-microg rhBMP-2 groups at 4 weeks compared to unfilled controls, PLA only, and 17-microg rhBMP-2 groups and equivalent to the autograft. At 8 weeks all groups receiving rhBMP-2 were equivalent to the autograft and significantly greater than unfilled defects and PLA alone. Similarly, the biomechanical analysis indicated significantly greater torque at failure for the 35-microg rhBMP-2 group compared to all other groups at 4 weeks. By 8 weeks all groups receiving rhBMP-2 and autograft had significantly greater torque than unfilled controls and PLA alone. These radiomorphometric and biomechanical results indicate PLA may be a suitable carrier for rhBMP-2 used for skeletal regeneration.

Assessment of an experimental bone wax polymer plus TGF‐β1 implanted into calvarial defects

The study reported describes an experimental biodegradable polymer ceramic composite with wax-like handling properties that was combined with 2.0 micrograms of recombinant human transforming growth factor beta (rhTGF-beta(1)). The polymer/rhTGF-beta(1) combination was introduced into standard-sized calvarial defects in rabbits to evaluate biodegradability, biocompatibility, hemostasis control, and bone promotion. The experimental wound model was a standard-size circular calvarial defect 8 mm in diameter. The experimental design included 24 skeletally mature New Zealand white rabbits divided evenly between two time periods (6 and 12 weeks) and among three experimental treatments (untreated defects and defects treated with polymer with or without rhTGF-beta(1)). Evaluations consisted of clinical examinations, standarized radiography, radiomorphometry, as well as histology and histomorphometry. Data were analyzed by an Analysis of Variance (ANOVA) and Fishers Protected Least Significant Difference test at each time period (level of significance p < or = 0.05). Radiomorphometry data indicated that standard-sized defects treated with the wax-like polymer alone and the polymer plus 2.0 micrograms of TGF-beta(1) were significantly more radiopaque than control sites at both 6 and 12 weeks. Histomorphometric data revealed the amount of new bone was significantly greater at 6 weeks in the polymer plus 2.0 micrograms of TGF-beta(1) and in the control group than in the polymer alone. Moreover, at 12 weeks, there was significantly more new bone in the control than in either the polymer alone or the polymer plus 2.0 micrograms of TGF-beta(1). We speculate the incomplete biodegradation of the polymer ceramic composite contributed to the radiopacity and may have retarded osseous regeneration. It is important that the bone wax-like polymer material was biocompatible and acted as a hemostatic agent.